Microbial production of l-ascorbic acid

ABSTRACT

The present invention discloses an isolated polynucleotide molecule derived from a polynucleotide encoding a polypeptide having L-sorbosone dehydrogenase activity comprising a partial nucleotide sequence of at least 20 consecutive nucleotides of SEQ ID NO:1. The present invention further relates to a process for the production of L-ascorbic acid in high yield, in particular a process using resting cells of a microorganism able to convert given carbon sources into vitamin C. The thus obtained vitamin C may be further processed by purification and/or separation steps.

The present invention relates to polynucleotides derived frompolynucleotides which encode an enzyme which converts L-sorbosonedirectly to L-ascorbic acid. The enzyme L-sorbosone dehydrogenase (inthe following: SNDHai) produces L-ascorbic acid (vitamin C) directlyfrom L-sorbosone. The L-sorbosone dehydrogenase (SNDHai) was derivedfrom bacteria belonging to the genera Gluconobacter and Acetobacter. Thepresent invention further relates to a process for the production ofL-ascorbic acid in high yield. L-Ascorbic acid is widely used in thepharmaceutical, food and cosmetic industries.

For the past 70 years, L-ascorbic acid (vitamin C) has been producedindustrially from D-glucose by the well-known Reichstein method. Allsteps in this process are chemical except for one (the conversion ofD-sorbitol to L-sorbose) which is carried out by microbialtransformation. Since its initial implementation for industrialproduction of L-ascorbic acid, several chemical and technicalmodifications have been used to improve the efficiency of the Reichsteinmethod. Recent developments of vitamin C production are summarized inUllmann's Encyclopedia of Industrial Chemistry, 5^(th) Edition, Vol. A27(1996), pp. 547ff. Recently different steps of vitamin C production havebeen performed with the help of microorganisms or enzymes isolatedtherefrom.

Current production methods for L-ascorbic acid have some undesirablecharacteristics such as high-energy consumption and use of largequantities of organic and inorganic solvents. Therefore, over the pastdecades, other approaches to manufacture L-ascorbic acid using microbialconversions, which would be more economical as well as ecological, havebeen investigated. Direct L-ascorbic acid production has been reportedin several microorganisms.

Surprisingly, it has now been found that the direct conversion ofL-sorbosone to L-ascorbic acid can be performed using the L-sorbosonedehydrogenase (hereafter called SNDHai) isolated from G. oxydans N44-1or enzymes which are orthologs thereof originating from acetic acidbacteria belonging to the genera Gluconobacter and Acetobacter. A generesponsible for this reaction was isolated and the sequence wasdetermined. The sorbosone dehydrogenase enzyme encoded by this geneconverts L-sorbosone to L-ascorbic acid. This enzyme is different fromthe known SNDH enzymes. L-ascorbic acid or vitamin C as usedinterchangeably herein may be any chemical form of L-ascorbic acid foundin aqueous solutions, such as for instance undissociated, in its freeacid form or dissociated as an anion. The solubilized salt form ofL-ascorbic acid may be characterized as the anion in the presence of anykind of cations usually found in fermentation supernatants, such as forinstance potassium, sodium, ammonium, or calcium. Also included may beisolated crystals of the free acid form of L-ascorbic acid. On the otherhand, isolated crystals of a salt form of L-ascorbic acid are called bytheir corresponding salt name, i.e. sodium ascorbate, potassiumascorbate, calcium ascorbate and the like.

Conversion of L-sorbosone into vitamin C means that the conversion ofthe substrate resulting in vitamin C is performed by SNDHai, i.e. thesubstrate may be directly converted into vitamin C.

A cloning vector may be for instance any plasmid or phage DNA or otherDNA sequence which is able to replicate autonomously in a host cell, andwhich is characterized by one or a small number of restrictionendonuclease recognition sites at which such DNA sequences may be cut ina determinable fashion without loss of an essential biological functionof the vector, and into which a DNA fragment may be spliced in order tobring about its replication. The cloning vector may further contain forinstance a marker suitable for use in the identification of cellstransformed with the cloning vector. Such markers may provide forinstance resistance to antibiotics, such as for instance tetracycline orampicillin.

An expression vector may be any vector which is capable of enhancing theexpression of a gene that has been cloned into it, after for instancetransformation into a host. The cloned gene is usually placed under thecontrol of (i.e., operably linked to) certain control sequences such asfor instance promoter sequences. Promoter sequences may be eitherconstitutive or inducible.

A nucleic acid molecule may include DNA and RNA. Any form, such as forinstance double-stranded, single-stranded nucleic acid, and nucleosidesthereof may be useful as nucleic acid molecule. Also included arehybrids such as for instance DNA-RNA hybrids, DNA-RNA-protein hybrids,RNA-protein hybrids, and DNA-protein hybrids. A polynucleotide mayconsist of several bases, usually at least 20 nucleotide bases.

The term “homology” designates the similarity of two polynucleotidesequences. In order to determine the homology the polynucleotidesequences are arranged in such a manner, that similar areas can becompared. If required, nucleotides at certain positions may be replacedby a blank position in order to improve the similarity. Homologycomparisons may beperformed for instance by hand or by using computerprograms which are commercially available. Preferably the program is rununder standard conditions in order to obtain the maximum homology. Thedegree of homology or similarity between two nucleotide sequences isgiven in “% homology”.

A mutation may befor instance a single base pair change, insertion ordeletion in the nucleotide sequence of interest or a genetic event suchas an insertion of a genetic element like for instance a transposon.

The generation of a mutation into the DNA, i.e. mutagenesis, maybeperformed in different ways, such as for instance randomly, i.e.random mutagenesis, wherein the exact site of mutation is notpredictable, occurring anywhere in the chromosome(s) of themicroorganism or on the endogenous plasmid(s). The mutation may bealsogenerated as for instance a result of physical damage caused by agentssuch as for instance radiation, chemical treatment, or insertion of agenetic element.

As promoter may be used any DNA sequence which is located proximal tothe start codon of a respective gene and which initiates thetranscription of one or more adjacent gene(s). In general, the promotermay belocated at the 5′ region of a respective gene. The promoter may bean inducible or constitutive promoter. In the case of an induciblepromoter, the rate of transcription increases in response to an inducingagent. In case of a constitutive promoter, the rate of transcription isnot regulated by for instance an inducing agent.

The term “identity” and “% identity” refers to the comparison of twoamino acid sequences using a sequence analysis program as for instanceexemplified below. “% identical” refers to the percent of the aminoacids of the subject amino acid sequence that have been matched toidentical amino acids in the compared amino acid sequence. If both aminoacid sequences which are compared do not differ in any of their aminoacids, they are identical or have 100% identity.

In one embodiment the present invention is related to an isolatedpolynucleotide derivable from a polynucleotide molecule encoding apolypeptide having L-sorbosone dehydrogenase activity comprising apartial nucleotide sequence of SEQ ID NO:1 of at least 20 consecutivenucleotides. Thus, the present invention provides an isolatedpolynucleotide molecule derived from a polynucleotide encoding apolypeptide having L-sorbosone dehydrogenase activity comprising apartial nucleotide sequence of at least 20 consecutive nucleotides ofSEQ ID NO:1. The isolated polynucleotide comprises preferably a partialnucleotide sequence of at least 50 and more preferably of at least 100consecutive nucleotides of SEQ ID NO:1. Most preferred is an isolatedpolynucleotide comprising the nucleotide sequence of SEQ ID NO:1.Further most preferred embodiment is an isolated polynucleotidecomprising a nucleotide sequence selected from the group consisting ofSEQ ID NOs:11, 13, 15, 17, 19, 21 and 26. The isolated polynucleotidemay be derived from a polynucleotide which codes for a polypeptidehaving L-sorbosone dehydrogenase activity. SEQ ID NO:1 represents thecomplete nucleotide sequence of SNDHai which was isolated from themicroorganism Gluconobacter oxydans N44-1. Parts of such a sequence maybe used for different purposes. Short polynucleotides for example may beused as primers for, e.g., the amplification of suitable polynucleotidesisolated from other organisms. A short polynucleotide can be in therange of about 10 to about 100 base pairs (bp), usually about 14 toabout 50, and preferably about 17 to about 30 bp. Examples of such shortpolynucleotide sequences are represented by SEQ ID NOs:5, 6, 7, 8, 9,10, 23 or 24. Longer polynucleotides may code for polypeptides havingenzymatic activity. For example, the SNDHai has a transmembrane domainwhich may not be used for enzymatic activity. If parts of thepolynucleotide coding for the enzymatically active area of the proteinare expressed without the transmembrane domain, such a polypeptide mayhave sufficient enzymatic activity.

The isolated polynucleotide molecule is usually derived from a longerpolynucleotide sequence which codes also for a polypeptide havingL-sorbosone dehydrogenase activity. Such polynucleotides may be isolatedfor instance from bacteria. Preferably they are isolated from bacteriabelonging to the genera Gluconobacter and Acetobacter including, but notlimited to, G. oxydans, G. frateurii, G. cerinus and A. aceti. When suchpolynucleotides are derived from longer polynucleotide sequences it ispossible to determine the homology between such a polynucleotidesequence and SEQ ID NO:1. In such a case preferably an area having atleast 100 consecutive nucleotides is selected and the correspondingstretch from the other polynucleotide can be compared therewith. Whenthe polynucleotide sequence and the corresponding stretch derivable fromSEQ ID NO:1 have for instance 60 nucleotides which are identical bycomparing 100 consecutive nucleotides then the homology is 60%. Thus, inone embodiment the invention is directed to an isolated polynucleotideaccording to the present invention wherein the partial nucleotidesequence is derived from a polynucleotide sequence having a homology ofat least 60% with SEQ ID NO:1 whereby at least 100 consecutivenucleotides are compared. Preferably, the partial polynucleotidesequences of the present invention have a homology of at least 80% andmore preferably of at least 90% with SEQ ID NO:1. For the determinationof the homology stretches of for instance at least 100 may be used,preferably stretches of at least 300 and more preferably stretches of atleast 500 consecutive nucleotides.

The present invention provides novel polynucleotide sequences coding forL-sorbosone dehydrogenase of a microorganism belonging to acetic acidbacteria including the genera Gluconobacter and Acetobacter forproducing L-ascorbic acid from L-sorbosone. The said polynucleotidepreferably codes for a polypeptide having the amino acid sequence of SEQID NO:2 or a polypeptide derived or derivable from said polypeptide byfor instance substitution, deletion, insertion or addition of one ormore amino acids in the amino acid sequence of SEQ ID NO:2, whichretains L-sorbosone dehydrogenase activity to produce L-ascorbic acidfrom L-sorbosone. Further included are polynucleotide sequences codingfor partial polypeptide sequences of a polypeptide which retainsL-sorbosone dehydrogenase activity to produce L-ascorbic acid fromL-sorbosone such as, for example, polypeptides represented by SEQ IDNOs:12, 14, 16, 18, 20, 22, and 27.

The polypeptides of the present invention comprise preferably partialamino acid sequences of at least 25 consecutive amino acids selectedfrom the amino acids sequences of the polypeptides disclosed in thepresent application. The person skilled in the art is aware of the factthat certain stretches in polypeptides are essential for the biologicalactivity. There are, however, other areas wherein amino acids can beinserted, deleted or substituted by other amino acids preferably suchamino acids which are similar to the amino acids to be replaced.

This invention is further directed to recombinant DNA molecules and/orexpression vectors comprising a polynucleotide of the present invention,especially one which functions in a suitable host cell.

Any cell that serves as recipient of foreign nucleotide acid molecule(s)may beused as a host cell, such as for instance a cell carrying areplicable expression vector or cloning vector or a cell beinggenetically engineered by well known techniques to contain desiredgene(s) on its chromosome(s) or genome. The host cell may be ofprokaryotic or eukaryotic origin, such as, for instance bacterial cells,animal cells, including human cells, fungal cells, including yeastcells, and plant cells. Preferably the host cell belongs to bacteriathat can express the L-sorbosone dehydrogenase as an active form invivo, more preferably bacteria of the genera Gluconobacter, Acetobacter,Pseudomonas, such as P. putida, or Escherichia, such as E. coli.

Thus, it is an aspect of the present invention to provide a host cell asdescribed above, comprising such an expression vector or comprising sucha nucleotide which has the polynucleotide integrated in its chromosomalDNA. Such host cell is then called a recombinant host cell orrecombinant organism.

This invention is further directed to a process for producing arecombinant L-sorbosone dehydrogenase polypeptide encoded by apolynucleotide of this invention. Such process includes for instance thecultivation of any of the recombinant organisms of this invention asdescribed specifically above. Accordingly, part of this invention is therecombinant L-sorbosone dehydrogenase polypeptide produced by thisprocess. Such recombinant L-sorbosone dehydrogenase may be used forinstance as a soluble enzyme in any standard procedure used forenzymatic reactions and known to a skilled person, recycled by use ofdevices such as membrane modules or membrane reactors, or immobilized ona solid carrier for solid phase enzymatic reaction.

Another aspect of this invention is a process for producing L-ascorbicacid comprising converting a substrate into L-ascorbic acid with the aidof the recombinant L-sorbosone dehydrogenase polypeptide encoded by apolynucleotide of this invention. The use of an L-sorbosonedehydrogenase (SNDHai) isolated from a microorganism producing suchenzyme naturally, i.e., non-recombinantly, wherein the isolated SNDHaiis encoded by a polynucleotide of this invention, is also included bythe present invention.

As substrate may be used a carbon source that can be converted intoL-ascorbic acid by the SNDHai as encoded by a polynucleotide of thepresent invention. Preferred substrates are selected from L-sorbose,D-sorbitol, and L-sorbosone.

In one embodiment of this invention, the process for producingL-ascorbic acid comprises converting L-sorbose or D-sorbitol intoL-ascorbic acid in a host cell having the ability for convertingL-sorbose into L-sorbosone or for converting D-sorbitol intoL-sorbosone.

In another embodiment, the process for the production of L-ascorbic acidcomprises converting L-sorbosone into L-ascorbic acid with the aid ofthe recombinant SNDHai encoded by a polynucleotide of this invention.L-sorbosone dehydrogenase (SNDHai) isolated from a microorganismproducing such enzyme naturally, i.e., non-recombinantly, wherein theisolated SNDHai is encoded by a polynucleotide of this invention, may bealso used for such a process.

The invention provides isolated nucleic acid molecules encoding theenzyme (L-sorbosone dehydrogenase SNDHai or parts thereof). Methods andtechniques designed for the manipulation of isolated nucleic acidmolecules are well known in the art. Methods for the isolation,purification, and cloning of nucleic acid molecules, as well as methodsand techniques describing the use of eukaryotic and prokaryotic hostsand nucleic acid and protein expression therein, are known to theskilled person.

Functional derivatives of polypeptides of the present invention may bealso part of the present invention and are defined on the basis of theamino acid sequences of the present invention by addition, insertion,deletion and/or substitution of one or more amino acid residues of suchsequences wherein such derivatives preferably still have the L-sorbosonedehydrogenase activity measured by an assay known in the art orspecifically described herein. Such functional derivatives may bemadeeither by chemical peptide synthesis known in the art or by recombinanttechniques on the basis of the DNA sequences as disclosed herein bymethods known in the state of the art. Amino acid exchanges in proteinsand peptides which do not generally alter the activity of such moleculesare known.

In particular embodiments of the present invention, conservativesubstitutions of interest occur as follows: as example substitutions,Ala to Val/Leu/Ile, Arg to Lys/Gln/Asn, Asn to Gln/His/Lys/Arg, Asp toGlu, Cys to Ser, Gln to Asn, Glu to Asp, Gly to Pro/Ala, His toAsn/Gln/Lys/Arg, Ile to Leu/Val/Met/Ala/Phe/norLeu, Lys to Arg/Gln/Asn,Met to Leu/Phe/Ile, Phe to Leu/Val/Ile/Ala/Tyr, Pro to Ala, Ser to Thr,Thr to Ser, Trp to Tyr/Phe, Tyr to Trp/Phe/Thr/Ser, and Val toIle/Leu/Met/Phe/Ala/norLeu are reasonable. As preferred examples, Ala toVal, Arg to Lys, Asn to Gln, Asp to Glu, Cys to Ser, Gln to Asn, Glu tAsp, Gly to Ala, His to Arg, Ile to Leu, Leu to Ile, Lys to Arg, Met toLeu, Phe to Leu, Pro to Ala, Ser to Thr, Thr to Ser, Trp to Tyr, Tyr toPhe, and Val to Leu are reasonable. If such substitutions result in achange in biological activity, then more substantial changes,denominated exemplary substitutions described above, are introduced andthe products screened.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the model Applied Biosystems PRISM 310 geneticanalyzer). Therefore, as is known in the art for any DNA sequencedetermined by this automated approach, any nucleotide sequencedetermined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% homologous,more typically at least about 95% to at least about 99.9% homologous tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

In a preferred embodiment the present invention is directed topolynucleotides encoding polypeptide having the L-sorbosonedehydrogenase activity as disclosed in the sequence listing as SEQ IDNO:2 as well as the complementary strands, or those which include thesesequences, DNA sequences or fragments thereof, and DNA sequences, whichhybridize under standard conditions with such sequences but which encodefor polypeptides having exactly the same amino acid sequence.

Another mode of describing the similarity of polynucleotide sequences isto determine whether such sequences do hybridize or do not hybridize.This depends on the conditions selected for the hybridization.

Standard conditions for hybridization mean in this context suchconditions which are generally used by a person skilled in the art todetect specific hybridization signals, or preferably so called“stringent hybridization conditions” used by a person skilled in theart. Thus, as used herein, the term “stringent hybridization conditions”means that hybridization will occur if there is about 95% and preferablyat least 97% homology between the sequences. Stringent hybridizationconditions are, e.g., 2 h to 4 days incubation at 42° C. using adigoxigenin (DIG)-labeled DNA probe (prepared by using a DIG labelingsystem; Roche Diagnostics GmbH, 68298 Mannheim, Germany) in a solutionsuch as DigEasyHyb solution (Roche Diagnostics GmbH) with or without 100μg/ml salmon sperm DNA, or a solution comprising 50% formamide, 5×SSC(150 mM NaCl, 15 mM trisodium citrate), 0.02% sodium dodecyl sulfate,0.1% N-lauroylsarcosine, and 2% blocking reagent (Roche DiagnosticsGmbH), followed by washing the filters twice for 5 to 15 minutes in2×SSC and 0.1% SDS at room temperature and then washing twice for 15-30minutes in 0.5×SSC and 0.1% SDS or 0.1×SSC and 0.1% SDS at 65-68° C.

In one aspect, the gene encoding L-sorbosone dehydrogenase, the nucleicacid molecule containing said gene, the expression vector and therecombinant organism used in the present invention may be obtained bythe following steps:

(1) transposon mutagenesis as described below on the strains belongingto the genus Gluconobacter or Acetobacter strains that producesL-ascorbic acid from L-sorbosone to obtain colonies expressingantibiotic resistance encoded by the transposon used;

(2) selection of L-ascorbic acid non-producing mutants in the screeningwith L-sorbosone as a substrate;

(3) isolation of chromosomal DNA from the mutants;

(4) cloning of the DNA fragment containing the transposon from thechromosomal DNA by colony-, plaque-, or Southern-hybridization, PCR(polymerase chain reaction) cloning, and so on;

(5) determination of the nucleotide sequence of the DNA fragmentcontaining the transposon insertion;

(6) cloning of the DNA fragment from the parent strain that producesL-ascorbic acid from L-sorbosone;

(7) construction of the expression vector on which the gene coding forL-sorbosone dehydrogenase can express efficiently;

(8) construction of recombinant organisms carrying the gene coding forL-sorbosone dehydrogenase by an appropriate method for introducing DNAinto host cell, e.g. transformation, transduction, conjugal transferand/or electroporation, which host cell thereby becomes a recombinantorganism of this invention.

Transposon mutagenesis is known as a potent tool for genetic analysis(P. Gerhardt et al., “Methods for General and Molecular Bacteriology”Chapter 17, Transposon Mutagenesis; American Society for Microbiology).

A variety of transposons is known in the art, such as Tn3, Tn5, Tn7,Tn9, Tn10, phage Mu and the like. Among them, Tn5 is known to havealmost no insertion specificity, and its size is relatively small. Forthe purpose of use in the random mutagenesis in the practice of thepresent invention, Tn5 is preferred. A variety of Tn5 derivatives,designated Mini-Tn5s, which consist of 19 by of the Tn5 inverted repeatsrequired for transposition coupled to antibiotic resistance or otherselectable marker genes are also useful for the present invention. SuchMini-Tn5s are inserted into a suicide vector, in addition to the Tn5transposase (tnp), to construct an efficient suicide Tn5 mutagenesissystem.

Random mutagenesis with transposon involves the introduction of atransposon into a target bacterial cell via for instance transformation,transduction, conjugal mating or electroporation by using suicideplasmid or phage vectors. The resulting mutants may be screened with theaid of the marker carried by the transposon. Transposition of thetransposon into the genome of the recipient bacterium may bedetectedafter the vector used has been lost by segregation.

For the introduction of transposons into a microorganisms of the genusGluconobacter or Acetobacter, so-called suicide vectors including forinstance a derivative of phage P1 and narrow-host-range plasmids such asa derivative of pBR325 carrying ColEl replication origin are commonlyused. The phage P1 vectors and the plasmid vectors may be transferred byinfection and by transformation, conjugal mating or electroporation,respectively, into the recipient cells, wherein these vectors preferablylack the appropriate origins of recipients. The choice of suicide vectorand transposon to be used depends on criteria including for instancephage sensitivity, intrinsic antibiotic resistance of the recipientcell, the availability of a gene transfer system includingtransformation, conjugal transfer, electroporation, or infection tointroduce transposon-carrying vector into E. coli.

One of the preferable vectors for use in the present invention is forinstance phage P1 (ATCC25404) which injects its DNA into a microorganismbelonging to the genus Gluconobacter or Acetobacter, however, this DNAwill be unable to replicate and will be lost by segregation. Such P1phage carrying Tn5 (P1::Tn5) may be used in the form of phage lysateswhich may be prepared by lysing E. coli carrying P1::Tn5 in accordancewith known procedures (see: e.g. “Methods for General and MolecularBacteriology” Chapter 17, Transposon Mutagenesis; American Society forMicrobiology or US patent 5082785, 1992).

For confirming that the deficient mutant really carries the transposon,methods such as for instance colony- or Southern-hybridization may beconducted with labeled-DNA fragments containing the transposon used asthe probe by standard methods (Molecular cloning, a laboratory manualsecond edition, Maniatis T., et al., 1989).

Such a mutant was isolated as described in Example 5 of the presentinvention. The transposon mutant may be useful for further identifyingthe target gene coding for L-sorbosone dehydrogenase and determining thenucleotide sequence of the region tagged with the transposon.

The DNA fragment containing a transposon insertion may be cloned intoany E. coli cloning vectors, preferably pUC18, pUC19, pBluescript IIKS+(Stratagene Europe) and their relatives, by selecting transformantsshowing both phenotypes of selection markers of the vector and thetransposon. The nucleotide sequences adjacent to the transposon may bedetermined by sequencing methods known in the art.

Alternatively, when the said L-sorbosone dehydrogenase polypeptide ispurified from the strain producing L-ascorbic acid from L-sorbosone, thedesired gene may be cloned in either plasmid or phage vectors from atotal chromosomal DNA by the following illustrative methods:

(i) The partial amino acid sequences can be determined from the purifiedproteins or peptide fragments thereof by methods such as, for instance,matrix assisted laser desorption/ionization (MALDI). Such whole proteinor peptide fragments may be prepared by the isolation of such a wholeprotein or by peptidase-treatment from the gel after SDS-polyacrylamidegel electrophoresis (SDS-PAGE). Thus obtained protein or fragmentsthereof may also be applied to a protein sequencer such as AppliedBiosystems automatic gas-phase sequencer 470A. The amino acid sequencesmay be utilized to design and prepare oligonucleotide probes and/orprimers with DNA synthesizer such as for instance Applied Biosystemsautomatic DNA sequencer 381A. The probes may be used for isolatingclones carrying the target gene from a gene library of the straincarrying the target gene by means of for instance Southern-, colony- orplaque-hybridization.

(ii) Further alternatively, for the purpose of selecting clonesexpressing the target protein from the gene library, immunologicalmethods with for instance an antibody prepared against the targetprotein may be applied.

(iii) The DNA fragment of the target gene may be amplified from thetotal chromosomal DNA by for instance PCR with a set of primers, i.e.two oligonucleotides synthesized according to the amino acid sequencesdetermined as above. Then a clone carrying the whole gene may beisolated from the gene library constructed, e.g. in E. coli by forinstance Southern-, colony-, or plaque-hybridization with the PCRproduct obtained above as the probe.

DNA sequences which may bemade by PCR using primers designed on thebasis of the DNA sequences disclosed therein by methods known in the artare also an object of the present invention.

The above-mentioned antibody may be prepared for instance with thepurified L-sorbosone dehydrogenase proteins, the purified recombinantL-sorbosone dehydrogenase proteins such as for instance His-taggedL-sorbosone dehydrogenase expressed in E. coli, or its peptide fragmentas an antigen. A polypeptide sequence deduced from a nucleotide sequenceof the L-sorbosone dehydrogenase may be used as an antigen forpreparation of an antibody.

Once a clone carrying the desired gene is obtained, the nucleotidesequence of the target gene may be determined by a well-known method.

To express the desired gene/nucleotide sequence efficiently, variouspromoters may be used; e.g., the original promoter of the gene,promoters of antibiotic resistance genes such as for instance kanamycinresistant gene of Tn5, ampicillin resistant gene of pBR322, andbeta-galactosidase of E. coli (lac), trp-, tac-, trc-promoter, promotersof lambda phage and any promoters which may be functional in the hostcell. For this purpose, the host cell may be selected from a groupconsisting of bacterial cells, animal cells, including human cells,fungal cells, including yeast cells, and plant cells. Preferably thehost cell belongs to bacteria that can express the L-sorbosonedehydrogenase as an active form in vivo, in particular bacteria of thegenera Gluconobacter, Acetobacter, Pseudomonas and Escherichia.

For expression, other regulatory elements, such as for instance aShine-Dalgarno (SD) sequence (e.g., AGGAGG and so on including naturaland synthetic sequences operable in the host cell) and a transcriptionalterminator (inverted repeat structure including any natural andsynthetic sequence) which are operable in the host cell (into which thecoding sequence will be introduced to provide a recombinant cell of thisinvention) may be used with the above described promoters.

A wide variety of host/cloning vector combinations may be employed incloning the double stranded DNA. Preferred vectors for the expression ofthe gene of the present invention, i.e. the SNDHai gene, in E. coli maybe selected from any vectors usually used in E. coli, such as forinstance pQE vectors which can express His-tagged recombinant proteins(QIAGEN AG Switzerland), pBR322 or its derivatives including forinstance pUC18 and pBluescript II (Stratagene Cloning Systems, Calif.,USA), pACYC177 and pACYC184 and their derivatives, and a vector derivedfrom a broad host range plasmid such as RK2 and RSF 1010. A preferredvector for the expression of the nucleotide sequence of the presentinvention in bacteria including Gluconobacter, Acetobacter, andPseudomonas is selected from any vectors which can replicate inGluconobacter, Acetobacter, or Pseudomonas as well as in a preferredcloning organism such as E. coli. The preferred vector is abroad-host-range vector such as for instance a cosmid vector like pVK100and its derivatives and RSF1010. Copy number and stability of the vectorshould be carefully considered for stable and efficient expression ofthe cloned gene and also for efficient cultivation of the host cellcarrying the cloned gene. Nucleic acid molecules containing for instancetransposable elements such as Tn5 may also be used as a vector tointroduce the desired gene into the preferred host, especially on achromosome. Nucleic acid molecules containing any DNAs isolated from thepreferred host together with the SNDHai gene of the present inventionmay be also useful to introduce this gene into the preferred host cell,especially on a chromosome. Such nucleic acid molecules maybetransferred to the preferred host by applying any of a conventionalmethods, e.g., transformation, transduction, conjugal mating orelectroporation, which are well known in the art, considering the natureof the host cell and the nucleic acid molecule.

The L-sorbosone dehydrogenase gene/nucleotide sequences provided in thisinvention may be ligated into a suitable vector containing a regulatoryregion such as for instance a promoter, a ribosomal binding site, and atranscriptional terminator operable in the host cell described abovewith a well-known method in the art to produce an expression vector.

To construct a recombinant microorganism carrying an expression vector,various gene transfer methods including for instance transformation,transduction, conjugal mating, and electroporation may be used. Themethod for constructing a recombinant cell may be selected from themethods well-known in the field of molecular biology. For instance,conventional transformation systems may be used for Gluconobacter,Acetobacter, Pseudomonas, or Escherichia. A transduction system may alsobe used for E. coli. Conjugal mating system may be widely used inGram-positive and Gram-negative bacteria including for instance E. coli,P. putida, and Gluconobacter. An example of conjugal mating is disclosedin WO 89/06,688. The conjugation may occur in for instance liquid mediumor on a solid surface. Examples for a recipient for SNDHai productioninclude for instance microorganisms of Gluconobacter, Acetobacter,Pseudomonas, or Escherichia. To the recipient for conjugal mating, aselective marker may be added; e.g., resistance against nalidixic acidor rifampicin is usually selected. Natural resistance may also be used,e.g., resistance against polymyxin B is useful for many Gluconobacters.

The present invention provides recombinant L-sorbosone dehydrogenase(SNDHai). One may increase the production yield of the L-sorbosonedehydrogenase enzyme by introducing the L-sorbosone dehydrogenase geneprovided by the present invention into host cells described above. Inone aspect, the L-sorbosone dehydrogenase proteins are produced in ahost cell selected from a group consisting of Gluconobacter,Acetobacter, Pseudomonas, or Escherichia by using the L-sorbosonedehydrogenase gene of the present invention.

The microorganism which is able to express SNDHai as encoded by apolynucleotide sequence of the present invention may be cultured in anaqueous medium supplemented with appropriate nutrients under aerobicconditions. The cultivation may be conducted in batch, fed-batch,semi-continuous or continuous mode. The cultivation period may varydepending on for instance the host used for expression of the targetpolypeptide, pH, temperature and nutrient medium to be used, and ispreferably about 1 to about 10 days when run in batch or fed-batch mode.The cultivation may be conducted at for instance a pH of about 4.0 toabout 9.0, preferably about 5.0 to about 8.0. The preferred temperaturerange for carrying out the cultivation is from about 13° C. to about 36°C., preferably from about 18° C. to about 33° C. Usually, the culturemedium may contain such nutrients as assimilable carbon sources, e.g.,glycerol, D-mannitol, D-sorbitol, L-sorbose, erythritol, ribitol,xylitol, arabitol, inositol, dulcitol, D-ribose, D-fructose, D-glucose,and sucrose, preferably D-sorbitol, D-mannitol, and glycerol; anddigestible nitrogen sources such as organic substances, e.g., peptone,yeast extract, baker's yeast, urea, amino acids, and corn steep liquor.Various inorganic substances may also be used as nitrogen sources, e.g.,nitrates and ammonium salts. Furthermore, the culture medium usually maycontain inorganic salts, e.g., magnesium sulfate, manganese sulfate,potassium phosphate, and calcium carbonate.

It is understood that the process for the production of vitamin C fromthe substrate using a host comprising the SNDHai encoded by apolynucleotide as of the present invention is performed with growingcells, i.e., specific growth rates of the cells which are for instanceat least 0.02 h⁻¹.

One embodiment of the present invention is the use of isolated SNDHaiencoded by a nucleotide sequence as disclosed herein for the productionof vitamin C. For isolation and purification of SNDHai from themicroorganism after cultivation, the cells of the microorganism may beharvested from the liquid culture broth by for instance centrifugationor filtration. The harvested cells may be washed for instance withwater, physiological saline or a buffer solution having an appropriatepH. The washed cells may be suspended in an appropriate buffer solutionand disrupted by means of for instance a homogenizer, sonicator, Frenchpress, or by treatment with lysozyme and the like to give a solution ofdisrupted cells. The L-sorbosone dehydrogenase may be isolated andpurified from the cell-free extract or disrupted cells, preferably fromthe membrane fraction by standard methods such as for instanceultracentrifugation, differential solubilization using appropriatedetergents, precipitation by salts or other suitable agents, dialysis,ion exchange chromatographies, hydroxyapatite chromatographies,hydrophobic chromatographies, size exclusion chromatographies, affinitychromatographies, or crystallization. When the recombinant L-sorbosonedehydrogenase is produced as tagged polypeptide such as for instance aHis-tag one, it may be purified with affinity resins such as forinstance Nickel affinity resin. The purification of L-sorbosonedehydrogenase may be monitored photometrically by using for instanceartificial electron acceptors such as nitrobluetetrazolium chloride(NBT) and phenazine methosulfate, 2,6-dichlorophenol indophenole (DCIP),ferricyanide or cytochrome c.

The production of L-ascorbic acid with the help of the SNDHai asdescribed herein is provided by the present invention. The source forthe SNDHai is not critical. This process may be performed by using forinstance microorganisms able to naturally express active SNDHai enzyme,SNDHai encoded by a nucleotide sequence of the present invention whichis isolated from said microorganisms, recombinant organisms as describedabove carrying the SNDHai gene of the present invention, or by using thenative and/or recombinant SNDHai in the form of a membrane fraction,soluble or immobilized enzyme acting as a biocatalyst to convertL-sorbosone into L-ascorbic acid as described below. The above describedmethod for the isolation and purification of SNDHai may beused for boththe native and recombinant SNDHai.

Recombinant organisms used for the production of L-ascorbic acid fromL-sorbosone may be cultivated as described above. Preferably, therecombinant organism is selected from the group consisting ofGluconobacter, Acetobacter, Pseudomonas, and Escherichia carrying theL-sorbosone dehydrogenase gene of the present invention. The recombinantmicroorganism may be cultured under the same conditions as describedabove. If the recombinant organism being used for production ofL-ascorbic acid is not able to convert any of the carbon sourcesdescribed above into L-sorbosone, then L-sorbosone has to be added tothe medium to be used as the precursor for the production of L-ascorbicacid. The reaction period may vary depending on the pH, temperature andreaction mixture to be used, and is preferably about 1 to about 10 dayswhen run in batch or fed-batch mode.

In one embodiment, the SNDHai of the present invention, either asrecombinant or native, i.e. isolated non-recombinant enzyme, is purifiedfrom the culture medium as described above and used in the form ofsoluble or immobilized enzyme as a biocatalyst to convert L-sorbosoneinto L-ascorbic acid in any process mode known to the skilled person,such as batch, fed-batch, semi-continuous or continuous mode. Thepurified L-sorbosone dehydrogenase may be used for instance as such insoluble form, retained in the reaction vessel by means of membranedevices, or as immobilized enzyme in any solid phase such as forinstance porous or polymeric matrixes. For instance, the enzyme may bebound directly to a membrane, granules or the like of a resin having oneor more functional groups, or it may be bound to the resin throughbridging compounds having one or more functional groups, for example,glutaraldehyde. The reaction using purified enzyme in soluble, retainedor immobilized form may take place for instance in aqueous mediumcontaining L-sorbosone and other appropriate nutrients under aerobicconditions. The reaction medium may contain for instance inorganicsalts, e.g., magnesium sulfate, potassium phosphate, and calciumcarbonate. The reaction may be conducted at a pH of about 4.0 to about9.0, preferably about 5.0 to about 8.0. The preferred temperature rangefor carrying out the reaction is from about 13° C. to about 36° C.,preferably from about 18° C. to about 33° C.

Microorganisms which can be used for the present invention may bepublicly available from different sources, e.g., Deutsche Sammlung vonMikroorganismen and Zellkulturen (DSMZ), Mascheroder Weg 1B, D-38124Braunschweig, Germany, American Type Culture Collection (ATCC), P.O. Box1549, Manassas, Va. 20108 USA on May 12, 2003 or Culture CollectionDivision, NITE Biological Resource Center, 2-5-8, Kazusakamatari,Kisarazu-shi, Chiba, 292-0818, Japan (formerly: Institute forFermentation, Osaka (IFO), 17-85, Juso-honmachi 2-chome,Yodogawa-ku,Osaka 532-8686, Japan). Examples of preferred bacteria deposited withIFO are for instance Gluconobacter oxydans (formerly known as G.melanogenus) IFO 3293, Gluconobacter oxydans (formerly known as G.melanogenus) IFO 3292, Gluconobacter oxydans (formerly known as G.rubiginosus) IFO 3244, Gluconobacter frateurii (formerly known as G.industrius) IFO 3260, Gluconobacter cerinus IFO 3266, Gluconobacteroxydans IFO 3287,and Acetobacter aceti subsp. orleanus IFO 3259, whichwere all deposited on Apr. 5, 1954; Acetobacter aceti subsp. xylinum IFO13693 deposited on Oct. 22, 1975, and Acetobacter aceti subsp. xylinumIFO 13773 deposited on Dec. 8, 1977. Strain Acetobacter sp. ATCC 15164,which is also an example of a preferred bacterium, was deposited withATCC. Strain Gluconobacter oxydans (formerly known as G. melanogenus)N44-1 as another example of a preferred bacterium is a derivative of thestrain IFO 3293 and is described in Sugisawa et al., Agric. Biol. Chem.54: 1201-1209, 1990.

It is understood that the above-mentioned microorganisms also includesynonyms or basonyms of such species having the same physiologicalproperties, as defined by the International Code of Nomenclature ofProkaryotes.

In a further aspect, the present invention relates to a novel processfor the production of L-ascorbic acid (vitamin C) in high yield by usingresting cells of a microorganism able to convert given carbon sourcesinto vitamin C.

Direct L-ascorbic acid production has been reported in severalmicroorganisms, using different cultivation methods. The disadvantage ofsuch processes, however, is the low yield of vitamin C produced due tothe instability of the product. Using, for instance, microorganismswhich are known to be both capable of the production of 2-keto-L-gulonicacid (2-KGA) and vitamin C, the yield of microbiologically producedvitamin C is further limited by the relatively high production of 2-KGAwhich is more readily synthesized by said microorganism, leading, forinstance, to ratios between the concentration of vitamin C and 2-KGAwhich are less than 0.1. Thus, it is an object of the present inventionto improve the microbiological production of vitamin C to get higheryields as with the processes described in the prior art.

Surprisingly, it has now been found that a process using resting cellsof a microorganism capable of performing the direct conversion of asubstrate to vitamin C leads to higher yields of vitamin C.

In particular, the present invention provides a process for theproduction of vitamin C comprising converting a substrate into vitamin Cin a medium comprising resting cells of a microorganism.

As substrate for the above process using resting cells of amicroorganism may be used a carbon source that can be converted intoL-ascorbic acid and which is easily obtainable from the D-glucose orD-sorbitol metabolisation pathway such as, for example, D-glucose,D-sorbitol, L-sorbose, L-sorbosone, 2-keto-L-gulonate, D-gluconate,2-keto-D-gluconate or 2,5-diketo-gluconate. A further possible substratemight be galactose. Preferably, the substrate is selected from forinstance D-glucose, D-sorbitol, L-sorbose or L-sorbosone, morepreferably from D-glucose, D-sorbitol or L-sorbose, and most preferablyfrom D-sorbitol or L-sorbose. The term “substrate” and “productionsubstrate” in connection with the above process using resting cells of amicroorganism is used interchangeably herein.

Conversion of the substrate into vitamin C in connection with the aboveprocess using resting cells of a microorganism means that the conversionof the substrate resulting in vitamin C is performed by themicroorganism, i.e. the substrate may be directly converted into vitaminC. Said microorganism is cultured under conditions which allow suchconversion from the substrate as defined above.

A medium as used herein for the above process using resting cells of amicroorganism may be any suitable medium for the production of vitaminC. Typically, the medium is an aqueous medium comprising for instancesalts, substrate(s), and a certain pH. The medium in which the substrateis converted into vitamin C is also referred to as the productionmedium.

In connection with the above process using resting cells of amicroorganism any microorganism capable of performing the conversion ofthe substrate to vitamin C may be used, such as for instance, yeast,algae or bacteria, either as wild type strains, mutant strains derivedby classic mutagenesis and selection methods or as recombinant strains.Examples of such yeast may be, e.g., Candida, Saccharomyces,Zygosaccharomyces, Scyzosaccharomyces, or Kluyveromyces. An example ofsuch algae may be, e.g., Chlorella. Examples of such bacteria may be,e.g., Gluconobacter, Acetobacter, Ketogulonicigenium, Pantoea,Cryptococcus, Pseudomonas, such as, e.g., Pseudomonas putida, andEscherichia, such as, e.g., Escherichia coli. Preferred areGluconobacter or Acetobacter aceti, such as for instance G. oxydans, G.cerinus, G. frateurii, A. aceti subsp. xylinum or A. aceti subsp.orleanus.

In connection with the above process using resting cells of amicroorganism the microorganisms which can be used for the presentinvention may be publicly available from different sources, e.g.,Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ),Mascheroder Weg 1B, D-38124 Braunschweig, Germany, American Type CultureCollection (ATCC), P.O. Box 1549, Manassas, Va. 20108 USA on May 12,2003 or Culture Collection Division, NITE Biological Resource Center,2-5-8, Kazusakamatari, Kisarazu-shi, Chiba, 292-0818, Japan (formerly:Institute for Fermentation, Osaka (IFO), 17-85, Juso-honmachi2-chome,Yodogawa-ku, Osaka 532-8686, Japan). Examples of preferredbacteria deposited with IFO are for instance Gluconobacter oxydans(formerly known as G. melanogenus) IFO 3293, Gluconobacter oxydans(formerly known as G. melanogenus) IFO 3292, Gluconobacter oxydans(formerly known as G. rubiginosus) IFO 3244, Gluconobacter frateurii(formerly known as G. industrius) IFO 3260, Gluconobacter cerinus IFO3266, Gluconobacter oxydans IFO 3287,and Acetobacter aceti subsp.orleanus IFO 3259, which were all deposited on Apr. 5, 1954; Acetobacteraceti subsp. xylinum IFO 13693 deposited on Oct. 22, 1975, andAcetobacter aceti subsp. xylinum IFO 13773 deposited on Dec. 8, 1977.Strain Acetobacter sp. ATCC 15164, which is also an example of apreferred bacterium, was deposited with ATCC. Strain Gluconobacteroxydans (formerly known as G. melanogenus) N44-1 as another example of apreferred bacterium is a derivative of the strain IFO 3293 and isdescribed in Sugisawa et al., Agric. Biol. Chem. 54: 1201-1209, 1990.

In connection with the above process using resting cells of amicroorganism it is understood that the above-mentioned microorganismsalso include synonyms or basonyms of such species having the samephysiological properties, as defined by the International Code ofNomenclature of Prokaryotes. The nomenclature of the microorganisms asused herein is the one officially accepted (at the filing date of thepriority application) by the International Committee on Systematics ofProkaryotes and the Bacteriology and Applied Microbiology Division ofthe International Union of Microbiological Societies, and published byits official publication vehicle International Journal of Systematic andEvolutionary Microbiology (IJSEM). A particular reference is made toUrbance et al., IJSEM (2001) vol 51:1059-1070, with a correctivenotification on IJSEM (2001) vol 51:1231-1233, describing thetaxonomically reclassification of G. oxydans DSM 4025 asKetogulonicigenium vulgare.

As used herein, resting cells refer to cells of a microorganism whichare for instance viable but not actively growing, or which are growingat low specific growth rates [μ], for instance, growth rates that arelower than 0.02 h⁻¹, preferably lower than 0.01 h⁻¹. Cells which showthe above growth rates are said to be in a “resting cell mode”.

The process of the present invention as above using resting cells of amicroorganism may be performed in different steps or phases: preferably,the microorganism is cultured in a first step (also referred to as step(a) or growth phase) under conditions which enable growth. This phase isterminated by changing of the conditions such that the growth rate ofthe microorganism is reduced leading to resting cells, also referred toas step (b), followed by the production of vitamin C from the substrateusing the resting cells of (b), also referred to as production phase.

Growth and production phase as perfomed in the above process usingresting cells of a microorganism may be performed in the same vessel,i.e., only one vessel, or in two or more different vessels, with anoptional cell separation step between the two phases. The producedvitamin C can be recovered from the cells by any suitable means.Recovering means for instance that the produced vitamin C may beseparated from the production medium. Optionally, the thus producedvitamin C may be further processed.

For the purpose of the present invention relating to the above processusing resting cells of a microorganism, the terms “growth phase”,“growing step”, “growth step” and “growth period” are usedinterchangeably herein. The same applies for the terms “productionphase”, “production step”, “production period”.

One way of performing the above process using resting cells of amicroorganism as of the present invention may be a process wherein themicroorganism is grown in a first vessel, the so-called growth vessel,as a source for the resting cells, and at least part of the cells aretransferred to a second vessel, the so-called production vessel. Theconditions in the production vessel may be such that the cellstransferred from the growth vessel become resting cells as definedabove. Vitamin C is produced in the second vessel and recoveredtherefrom.

In connection with the above process using resting cells of amicroorganism, in one aspect, the growing step can be performed in anaqueous medium, i.e. the growth medium, supplemented with appropriatenutrients for growth under aerobic conditions. The cultivation may beconducted, for instance, in batch, fed-batch, semi-continuous orcontinuous mode. The cultivation period may vary depending on the kindof cells, pH, temperature and nutrient medium to be used, and may be forinstance about 10 h to about 10 days, preferably about 1 to about 10days, more preferably about 1 to about 5 days when run in batch orfed-batch mode, depending on the microorganism. If the cells are grownin continuous mode, the residence time may be for instance from about 2to about 100 h, preferably from about 2 to about 50 h, depending on themicroorganism. If the microorganism is selected from bacteria, thecultivation may be conducted for instance at a pH of about 3.0 to about9.0, preferably about 4.0 to about 9.0, more preferably about 4.0 toabout 8.0, even more preferably about 5.0 to about 8.0. If algae oryeast are used, the cultivation may be conducted, for instance, at a pHbelow about 7.0, preferably below about 6.0, more preferably below about5.5, and most preferably below about 5.0. A suitable temperature rangefor carrying out the cultivation using bacteria may be for instance fromabout 13° C. to about 40° C., preferably from about 18° C. to about 37°C., more preferably from about 13° C. to about 36° C., and mostpreferably from about 18° C. to about 33° C. If algae or yeast are used,a suitable temperature range for carrying out the cultivation may be forinstance from about 15° C. to about 40° C., preferably from about 20° C.to about 45° C., more preferably from about 25° C. to about 40° C., evenmore preferably from about 25° C. to about 38° C., and most preferablyfrom about 30° C. to about 38° C. The culture medium for growth usuallymay contain such nutrients as assimilable carbon sources, e.g.,glycerol, D-mannitol, D-sorbitol, L-sorbose, erythritol, ribitol,xylitol, arabitol, inositol, dulcitol, D-ribose, D-fructose, D-glucose,and sucrose, preferably L-sorbose, D-glucose, D-sorbitol, D-mannitol,and glycerol; and digestible nitrogen sources such as organicsubstances, e.g., peptone, yeast extract and amino acids. The media maybe with or without urea and/or corn steep liquor and/or baker's yeast.Various inorganic substances may also be used as nitrogen sources, e.g.,nitrates and ammonium salts. Furthermore, the growth medium usually maycontain inorganic salts, e.g., magnesium sulfate, manganese sulfate,potassium phosphate, and calcium carbonate.

In connection with the above process using resting cells of amicroorganism, in the growth phase the specific growth rates are forinstance at least 0.02 h⁻¹. For cells growing in batch, fed-batch orsemi-continuous mode, the growth rate depends on for instance thecomposition of the growth medium, pH, temperature, and the like. Ingeneral, the growth rates may be for instance in a range from about 0.05to about 0.2 h⁻¹, preferably from about 0.06 to about 0.15 h⁻¹, and mostpreferably from about 0.07 to about 0.13 h⁻¹.

In another aspect of the above process using resting cells of amicroorganism, resting cells may be provided by cultivation of therespective microorganism on agar plates thus serving as growth vessel,using essentially the same conditions, e.g., cultivation period, pH,temperature, nutrient medium as described above, with the addition ofagar agar.

In connection with the above process using resting cells of amicroorganism, if the growth and production phase are performed in twoseparate vessels, then the cells from the growth phase may be harvestedor concentrated and transferred to a second vessel, the so-calledproduction vessel. This vessel may contain an aqueous mediumsupplemented with any applicable production substrate that can beconverted to L-ascorbic acid by the cells. Cells from the growth vesselcan be harvested or concentrated by any suitable operation, such as forinstance centrifugation, membrane crossflow ultrafiltration ormicrofiltration, filtration, decantation, flocculation. The cells thusobtained may also be transferred to the production vessel in the form ofthe original broth from the growth vessel, without being harvested,concentrated or washed, i.e. in the form of a cell suspension. In apreferred embodiment, the cells are transferred from the growth vesselto the production vessel in the form of a cell suspension without anywashing or isolating step in-between.

Thus, in a preferred embodiment of the above process using resting cellsof a microorganism step (a) and (c) of the process of the presentinvention as described above are not separated by any washing and/orseparation step.

In connection with the above process using resting cells of amicroorganism, if the growth and production phase are performed in thesame vessel, cells may be grown under appropriate conditions to thedesired cell density followed by a replacement of the growth medium withthe production medium containing the production substrate. Suchreplacement maybe, for instance, the feeding of production medium to thevessel at the same time and rate as the withdrawal or harvesting ofsupernatant from the vessel. To keep the resting cells in the vessel,operations for cell recycling or retention may be used, such as forinstance cell recycling steps. Such recycling steps, for instance,include but are not limited to methods using centrifuges, filters,membrane crossflow microfiltration of ultrafiltration steps, membranereactors, flocculation, or cell immobilization in appropriate porous,non-porous or polymeric matrixes. After a transition phase, the vesselis brought to process conditions under which the cells are in a restingcell mode as defined above, and the production substrate is efficientlyconverted into vitamin C.

The aqueous medium in the production vessel as used for the productionstep in connection with the above process using resting cells of amicroorganism, hereinafter called production medium, may contain onlythe production substrate(s) to be converted into L-ascorbic acid, or maycontain for instance additional inorganic salts, e.g., sodium chloride,calcium chloride, magnesium sulfate, manganese sulfate, potassiumphosphate, calcium phosphate, and calcium carbonate. The productionmedium may also contain digestible nitrogen sources such as for instanceorganic substances, e.g., peptone, yeast extract, urea, amino acids, andcorn steep liquor, and inorganic substances, e.g. ammonia, ammoniumsulfate, and sodium nitrate, at such concentrations that the cells arekept in a resting cell mode as defined above. The medium may be with orwithout urea and/or corn steep liquor and/or baker's yeast. Theproduction step may be conducted for instance in batch, fed-batch,semi-continuous or continuous mode. In case of fed-batch,semi-continuous or continuous mode, both cells from the growth vesseland production medium can be fed continuously or intermittently to theproduction vessel at appropriate feed rates. Alternatively, onlyproduction medium may be fed continuously or intermittently to theproduction vessel, while the cells coming from the growth vessel aretransferred at once to the production vessel. The cells coming from thegrowth vessel may be used as a cell suspension within the productionvessel or may beused as for instance flocculated or immobilized cells inany solid phase such as porous or polymeric matrixes. The productionperiod, defined as the period elapsed between the entrance of thesubstrate into the production vessel and the harvest of the supernatantcontaining vitamin C, the so-called harvest stream, can vary dependingfor instance on the kind and concentration of cells, pH, temperature andnutrient medium to be used, and is preferably about 2 to about 100 h.The pH and temperature can be different from the pH and temperature ofthe growth step, but is essentially the same as for the growth step.

In a preferred embodiment of the above process using resting cells of amicroorganism, the production step is conducted in continuous mode,meaning that a first feed stream containing the cells from the growthvessel and a second feed stream containing the substrate is fedcontinuously or intermittently to the production vessel. The firststream may either contain only the cells isolated/separated from thegrowth medium or a cell suspension, coming directly from the growthstep, i.e. cells suspended in growth medium, without any intermediatestep of cell separation, washing and/or isolating. The second feedstream as herein defined may include all other feed streams necessaryfor the operation of the production step, e.g. the production mediumcomprising the substrate in the form of one or several differentstreams, water for dilution, and base for pH control.

In connection with the above process using resting cells of amicroorganism, when both streams are fed continuously, the ratio of thefeed rate of the first stream to feed rate of the second stream may varybetween about 0.01 and about 10, preferably between about 0.01 and about5, most preferably between about 0.02 and about 2. This ratio isdependent on the concentration of cells and substrate in the first andsecond stream, respectively.

Another way of performing the process as above using resting cells of amicroorganism of the present invention may be a process using a certaincell density of resting cells in the production vessel. The cell densityis measured as absorbance units (optical density) at 600 nm by methodsknown to the skilled person. In a preferred embodiment, the cell densityin the production step is at least about 10, more preferably betweenabout 10 and about 200, even more preferably between about 15 and about200, even more preferably between about 15 to about 120, and mostpreferably between about 20 and about 120.

In connection with the above process using resting cells of amicroorganism, in order to keep the cells in the production vessel atthe desired cell density during the production phase as performed, forinstance, in continuous or semi-continuous mode, any means known in theart may be used, such as for instance cell recycling by centrifugation,filtration, membrane crossflow ultrafiltration of microfiltration,decantation, flocculation, cell retention in the vessel by membranedevices or cell immobilization.

Further, in case the production step is performed in continuous orsemi-continuous mode and cells are continuously or intermittently fedfrom the growth vessel, the cell density in the production vessel may bekept at a constant level by, for instance, harvesting an amount of cellsfrom the production vessel corresponding to the amount of cells beingfed from the growth vessel.

In connection with the above process using resting cells of amicroorganism, the produced vitamin C contained in the so-called harveststream is recovered/harvested from the production vessel. The harveststream may include, for instance, cell-free or cell-containing aqueoussolution coming from the production vessel, which contains vitamin C asa result of the conversion of production substrate by the resting cellsin the production vessel. Cells still present in the harvest stream maybe separated from the vitamin C by any operations known in the art, suchas for instance filtration, centrifugation, decantation, membranecrossflow ultrafiltration or microfiltration, tangential flowultrafiltration or microfiltration or dead end filtration. After thiscell separation operation, the harvest stream is essentially free ofcells.

In connection with the above process using resting cells of amicroorganism, in one aspect, the process of the present invention leadsto yields of vitamin C which are at least about 1.8 g/l, preferably atleast about 2.5 g/l, more preferably at least about 4.0 g/l, and mostpreferably at least about 5.7 g/l. In one embodiment, the yield ofvitamin C produced by the process of the present invention is in therange of from about 1.8 to about 600 g/l. The yield of vitamin C refersto the concentration of vitamin C in the harvest stream coming directlyout of the production vessel, i.e. the cell-free supernatant comprisingthe vitamin C.

In connection with the above process using resting cells of amicroorganism, in one embodiment of the present invention, vitamin C isproduced by the process as described above using resting cells ofrecombinant microorganisms, such as for instance recombinant bacteria.Preferably, the recombinant bacteria are selected from bacteria that canexpress the L-sorbosone dehydrogenase as an active form in vivo, inparticular bacteria of the genera Gluconobacter, Acetobacter,Pseudomonas and Escherichia, most preferred from Gluconobacter,Acetobacter or E. coli. Even more preferred are for instance G. oxydansand E. coli and the most preferred is selected from the group consistingof G. oxydans N44-1, G. oxydans IFO 3293 and G. oxydans IFO 3244. Arecombinant microorganism may be any microorganism that is geneticallyengineered by well known techniques to contain one or more desiredgene(s) on its chromosome or on a plasmid introduced into saidmicroorganism, leading to, e.g., an overexpression of said gene(s). Thedesired gene(s) which are introduced into said microorganism may codefor instance for an enzyme involved in the conversion of a substrate tovitamin C. In a preferred embodiment, the desired gene encodes anL-sorbosone dehydrogenase, catalyzing the conversion of L-sorbosone tovitamin C. A preferred L-sorbosone dehydrogenase as used in the presentinvention is for instance the L-sorbosone dehydrogenase (SNDHai) of G.oxydans N44-1 (Sugisawa et al., Agric. Biol. Chem. 54: 1201-1209, 1990)as represented by SEQ ID NO:2, the nucleotide sequence encoding saidSNDHai is represented by SEQ ID NO:1. Functional derivatives of saidSNDHai can also be used for the purpose of the present invention. It isunderstood that nucleotide sequences having a homology of at least 80%,preferably of at least 90%, compared with SEQ ID NO:1 and which code forenzymes able to catalyze the conversion of L-sorbosone to vitamin C arealso part of the present invention.

The recombinant microorganism, such as for example G. oxydans N44-1, maycomprise several copies of SNDHai cloned on a suitable plasmid orintegrated on its chromosome. Plasmid copies which may be suitable forthe present invention are for instance in the range of about 2 to about15, preferably in the range of about 5 to about 10 per transformedmicroorganism. The number of plasmid copies may be determined by, forinstance, comparison of the intensity of a respective band visible onSDS-PAGE.

In connection with the above process using resting cells of amicroorganism, when using recombinant microorganisms for the process ofthe present invention, the growth and production step can be essentiallythe same as described above. If a recombinant microorganism comprisingSNDHai is used, such as for example recombinant G. oxydans N44-1 withincreased SNDHai dosage, the growth medium may contain for instanceD-sorbitol, L-sorbose, glycerol or D-glucose either alone or mixturesthereof, one or more suitable nitrogen sources and salts. The productionmedium may contain for instance D-sorbitol and/or L-sorbose and salts.Harvesting of vitamin C can be performed as essentially describedherein.

In a further aspect, the process of the present invention may becombined with further steps of separation and/or purification of theproduced vitamin C from other components contained in the harveststream, i.e., so-called downstream processing steps. These steps mayinclude any means known to a skilled person, such as, for instance,concentration, crystallization, precipitation, adsorption, ion exchange,electrodialysis, bipolar membrane electrodialysis and/or reverseosmosis. Vitamin C may be further purified as the free acid form or anyof its known salt forms by means of operations such as for instancetreatment with activated carbon, ion exchange, adsorption and elution,concentration, crystallization, filtration and drying. Specifically, afirst separation of vitamin C from other components in the harveststream might be performed by any suitable combination or repetition of,for instance, the following methods: two- or three-compartmentelectrodialysis, bipolar membrane electrodialysis, reverse osmosis oradsorption on, for instance, ion exchange resins or non-ionic resins. Ifthe resulting form of vitamin C is a salt of L-ascorbic acid, conversionof the salt form into the free acid form may be performed by forinstance bipolar membrane electrodialysis, ion exchange, simulatedmoving bed chromatographic techniques, and the like. Combination of thementioned steps, e.g., electrodialysis and bipolar membraneelectrodialysis into one step might be also used as well as combinationof the mentioned steps e.g. several steps of ion exchange by usingsimulated moving bed chromatographic methods. Any of these proceduresalone or in combination constitute a convenient means for isolating andpurifying the product, i.e. vitamin C. The product thus obtained mayfurther be isolated in a manner such as, e.g. by concentration,crystallization, precipitation, washing and drying of the crystalsand/or further purified by, for instance, treatment with activatedcarbon, ion exchange and/or re-crystallization.

In a preferred embodiment, vitamin C is purified from the harvest streamby a series of downstream processing steps as described above withouthaving to be transferred to a non-aqueous solution at any time of thisprocessing, i.e. all steps are performed in an aqueous environment. Suchpreferred downstream processing procedure may include for instance theconcentration of the harvest stream coming from the production vessel bymeans of two- or three-compartment electrodialysis, conversion ofvitamin C in its salt form present in the concentrated solution into itsacid form by means of bipolar membrane electrodialysis and/or ionexchange, purification by methods such as for instance treatment withactivated carbon, ion exchange or non-ionic resins, followed by afurther concentration step and crystallization. These crystals can beseparated, washed and dried. If necessary, the crystals may be againre-solubilized in water, treated with activated carbon and/or ionexchange resins and recrystallized. These crystals can then beseparated, washed and dried.

The following Examples further illustrate the present invention and arenot intended to limit the invention in any way.

EXAMPLE 1 L-Ascorbic Acid Production with Purified SNDHai

1. Purification of SNDHai:

Cells of a microorganism capable of producing SNDHai cultivated byfed-batch fermentation (for cultivation see Example 3) were suspended in25 ml of phosphate buffer (20 mM, pH 7.0) containing MgCl₂, 2 mM,dithiothreitol (DTT), 1 mM, and 2-3 EDTA-free protease inhibitor tablets(Roche Diagnostics GmbH). The cell suspension was treated three timeswith a French Pressure cell. Subsequently, 25 ml of phosphate buffer (20mM, pH 7.0) containing 2mM MgCl₂ and 1 M NaCl were added and thesuspension was ultracentrifuged (30.000 rpm, 60 min, 4° C.). The pelletcontaining the membrane fraction was washed with phosphate buffer (20mM, pH 7.0) containing 2 mM MgCl₂ and 500 mM NaCl and then suspended inan appropriate amount of phosphate buffer (20 mM, pH 7.0) containing 2mMMgCl₂. N-Octylglucoside (Fluka) was then added at a final concentrationof 2% (w/v) and the suspension was incubated for 90 min with gentlestirring on ice. After centrifugation (20.000 rpm, 60 min, 4° C.) theclear reddish supernatant was collected and polyethylene glycol 6000(Fluka) at a final concentration of 15% (w/v) was added. Afterincubation for 60 min at 4° C. with gentle shaking followed bycentrifugation (10.000 rpm, 30 min, 4° C.), the pellet was dissolved inTris-HCl buffer (20 mM, pH 7.6) containing 2 mM MgCl₂ and 0.5% (w/v)lauryl sulfobetaine (Fluka). After gentle shaking at 4° C. overnight thesolution was centrifuged (20.000 rpm, 30 min, 4° C.). The supernatantwas collected and further purified as follows.

The following purification steps were done at 4° C. on an AKTA Explorer10 S-system (Amersham Biosciences) with software UNICORN 3.1. Typicalflow rates for ion exchange chromatography were in the range of 1-2ml/min. Protein elution was monitored at 280 nm and SNDHai-activefractions were determined using the standard photometric assay at allstages of the purification (s. below) or the product assay with purifiedfractions.

The clear supernatant IV was desalted in 2.5 ml-portions on a Sephadex G25-gel filtration column (void volume: 2.5 ml) using 20 mM Tris-HClbuffer (pH 7.6) containing 2 mM MgCl₂ and 0.5% (w/v) laurylsulfobetaine.

SNDHai-positive fractions were pooled and an aliquot (approximately 10ml) was put on a strong anion exchange column (e.g. Mono-Q HR, AmershamBiosciences, column volume: 8 ml) which had been equilibrated prior touse with buffer A1 (10 mM Tris, 10 mM BisTris, 10 mM MES, 2 mM MgCl₂,0.5% lauryl sulfobetaine, pH 7.6). Non-binding proteins were eluted with100% buffer A1 and after 4 column volumes a linear pH-gradient in 6column volumes to 100% buffer B1 (Tris, 10 mM; BisTris, 10 mM; MES, 10mM; MgCl₂, 2 mM, and lauryl sulfobetaine, 0.5%, pH 4.7) was appliedfollowed by 8 column volumes of 100% buffer B1. SNDHai eluted at apH-value of approximately 6.5, which is very close to the pI-value of6.52 calculated from the amino acid sequence. Active fractions werepooled, diluted with the same amount of HEPES-buffer (50 mM, pH 8.0)containing 2 mM MgCl₂ and 0.5% lauryl sulfobetaine (final volume: 15-20ml), and applied to another strong anion exchange column (e.g. Mono-QHR, Amersham Biosciences, column volume: 1 ml) which had beenequilibrated with buffer A2 (15 mM HEPES, 2 mM MgCl₂, 0.5% laurylsulfobetaine, pH 7.6). Non-binding proteins were eluted with 100% bufferA2 and after 4 column volumes a linear salt-gradient in 20 columnvolumes to 40% buffer B2 (HEPES, 15 mM; MgCl₂, 2 mM, LiCl, 1 M, andlauryl sulfobetaine, 0.5%, pH 7.6) was applied followed by a stepgradient to 100% buffer B2. SNDHai eluted around 150 mM LiCl. Activefractions showed one single band at approximately 85 kDa in SDS gelelectrophoresis.

2. Photometric Assay for SNDHai.

The reaction mixture for the photometric SNDHai-activity measurementconsisted of 0.196 mM nitroblue tetrazolium chloride (NBT), 0.137 mMphenazine methosulfate (PMS), 20.4 mM L-sorbosone, and enzyme solutionin a final volume of 1.0 ml of 0.1 M sodium phosphate buffer, pH 7.5.The reaction was started with the addition of enzyme, and the enzymeactivity was measured in a cuvette with 1-cm light path as the initialreduction rate of NBT at 570 nm (T=25° C.). One unit of the enzymeactivity was defined as the amount of enzyme catalyzing the reduction of1 μM NBT per minute. The extinction coefficient of NBT at pH 7.5 wastaken as 100 mM⁻¹ cm⁻¹. Two kinds of reference cuvettes were used forthe activity determination: one contained the above-mentioned componentsexcept for L-sorbosone and another one contained all components exceptfor the enzyme solution.

3. Product Assay for SNDHai.

Pure SNDHai-containing fractions (see above) were analyzed directly forL-ascorbic acid production from L-sorbosone with an assay of thefollowing composition (0.5 ml total volume): 0.14 mg/ml of purified anddesalted SNDHai, 50 mM phosphate buffer (pH 6.5), 8 mg/ml bovine serumalbumin (BSA), 100 mM L-sorbosone, 1 mM PMS, 5 mM CaCl₂, 50 μM PQQ-K₂.The assay was conducted in appropriate reaction tubes at 25° C. withsufficient shaking (900 rpm on a benchtop shaker) under exclusion oflight.

Samples were analyzed by high performance liquid chromatography (HPLC)using an Agilent 1100 HPLC system (Agilent Technologies, Wilmington,USA) with a LiChrospher-100-RP18 (125×4.6 mm) column (Merck, Darmstadt,Germany) attached to an Aminex-HPX-78H (300×7.8 mm) column (Biorad,Reinach, Switzerland). The mobile phase was 0.004 M sulfuric acid, andthe flow rate was 0.6 ml/min. Two signals were recorded using a UVdetector (wavelength 254 nm) in combination with a refractive indexdetector. In addition, the identification of the L-ascorbic acid wasdone using an amino-column (YMC-Pack Polyamine-II, YMC, Inc., Kyoto,Japan) with UV detection at 254 nm. The mobile phase was 50 mM NH₄H₂PO₄and acetonitrile (40:60). Typical initial volumetric L-ascorbicacid-productivities under these conditions were 0.5-1.0 g/l/h. Thus,after 1 h reaction time, the concentration of L-ascorbic acid in thesupernatant was 300 to 930 mg/l.

EXAMPLE 2 L-Ascorbic Acid Production from L-Sorbose and D-Sorbitol inTube and Flask Fermentations

Cells of G. oxydans N44-1 were used to inoculate 4 ml of No. 3BD liquidmedium and cultivated in a tube (18 mm diameter) at 30° C. for 3 dayswith shaking at 220 rpm. 20 mg/l of L-ascorbic acid had accumulated atthe end of the incubation period.

Cells of strain N44-1 were cultivated (in triplicate) in 50 ml of No. 5medium containing 100 g/l D-sorbitol, 0.5 g/l glycerol, 15 g/l yeastextract (Difco), 2.5 g/l of MgSO₄.7H₂O, and 15 g/L of CaCO₃ in a 500 mlbaffled shake flask at 30° C. with shaking at 200 rpm. After 72 h ofcultivation, the amounts of L-ascorbic acid measured by HPLC in thethree flasks were 400, 500 and 680 mg/l.

EXAMPLE 3 L-ascorbic Acid Production from D-sorbitol in Fed-BatchFermentation

Cells of G. oxydans N44-1 were grown in 200 ml No. 5 medium containing100 g/l D-sorbitol, 0.5 g/l glycerol, 15 g/l yeast extract (FlukaBioChemika, Buchs, Switzerland), 2.5 g/l MgSO₄.7H₂O and 15 g/l CaCO₃ ina 2-l baffled shake flask at 30° C. with shaking at 180 rpm. After 48 h,150 ml of this culture was used to inoculate a 10-l bioreactor (B. Braun

ED10, Melsungen, Germany) previously prepared with 5.3 l mediumcontaining 20 g/l D-sorbitol, 0.5 g/l glycerol, 15 g/l yeast extract(Fluka BioChemika, Buchs, Switzerland) and 2.5 g/l MgSO₄.7H₂O andequipped with temperature, pH and dissolved oxygen sensors and controlloops. Temperature was controlled at 30° C., pH was controlled at 6.0 byadding a 28% ammonia solution, airflow was 4.5 l/min and dissolvedoxygen was controlled at 30% by a cascade with stirring speed (minimum300 rpm). After 6 h process time, a 500 g/l sorbitol solution was fed ata rate of 25 g/h for a period of 44 h. After 96 h process time, about 1%substrate was left in the supernatant, and 950 mg/l L-ascorbic acid hadbeen produced.

EXAMPLE 4 L-Ascorbic Acid Production from L-sorbosone or L-sorbose witha Cell Membrane Fraction

Cells of G. oxydans N44-1 were cultivated in 100 ml of No. 3BD liquidmedium in a 500 ml baffled shake flask at 30° C. with shaking at 220 rpmfor 3 days. The resulting culture was centrifuged at 500 rpm to removeCaCO₃. The supernatant from this step was then centrifuged at 5,000 rpmto pellet the cells. The collected cells were suspended in 3 ml of 50 mMpotassium phosphate buffer (pH7.0) and the cells were disrupted by twopassages through a French Pressure cell (SIM-AMINCO SpetronicInstruments, USA) at 900 psi. The resulting homogenate was firstcentrifuged at 5,000 rpm to remove cell debris. Then the supernatant wasdiluted to a final protein concentration of 3 mg of protein/ml. Thisdiluted sample is designated as cell-free extract (CFE). The CFE wascentrifuged at 100,000×g for 60 min. The supernatant was discarded andthe pellet was collected as the membrane fraction.

The reaction (200 μl) with the membrane fraction (100 μl) was carriedout in 50 mM potassium phosphate buffer (pH7.0), 30° C. with shaking at220 rpm for 15 h. The substrates tested were L-sorbosone (1% finalconcentration) and L-sorbose (2% final concentration). The final proteinconcentration used in the reaction was 1.5 mg/ml. At the end of theincubation period, 680 mg/l and 10 mg/l of L-ascorbic acid had beenproduced from 1% L-sorbosone and 2% L-sorbose, respectively.

EXAMPLE 5 Isolation of the SNDHai Gene from Gluconobacter oxydans N44-1

1. Tn5 Mutagenesis

Plasmid pSUP2021, a “suicide” vector containing Tn5 (Simon R. et al.1983. BIO/TECHNOLOGY 1: 784-791), was transferred from E. coli HB101into G. oxydans N44-1 by a conjugal mating method as follows. G. oxydansN44-1 was cultivated in a test tube containing 5 ml of MB liquid mediumat 30° C. overnight. E. coli HB101 carrying helper plasmid pRK2013 (D.H. Figurski, Proc. Natl. Acad. Sci. USA, 76, 1648-1652, 1979) and E.coli HB101 carrying plasmid pSUP2021 were cultivated in test tubescontaining 5 ml of LB medium with 50 μg/ml of kanamycin at 37° C.overnight. From the overnight cultures, cells of G. oxydans N44-1, E.coli HB101(pRK2013), and E. coli HB101(pSUP2021) were collectedseparately by centrifugation and suspended to the original volume in MBmedium. Then these cell suspensions were mixed in equal volumes and themixture was spread out on a 0.45 ρm nitrocellulose membrane laid on topof an MB agar plate. After cultivation at 27° C. for one day, the cellswere scraped off the membrane and dilutions were prepared in MB broth.The diluted cells were then spread on MB agar medium containing 10 μg/mlof polymixin B and 50 μg/ml of kanamycin (MPK medium). The polymixin Bselects against the E. coli donor and helper strains, while thekanamycin selects for those G. oxydans cells that have been transformedwith plasmid pSUP2021 (i.e., the transconjugants). About 30,000transconjugants were obtained.

2. Screening for L-ascorbic Acid Non-Producers.

In all, 3,760 transconjugants were transferred with sterile toothpicksonto MPK grid plates and grown at 27° C. for 3 days. To test forL-ascorbic acid production from L-sorbosone, cells of eachtransconjugant were picked off the grid plate with a sterile toothpickand suspended in 50 μl of a resting cell reaction mixture containing0.5% L-sorbosone, 0.3% NaCl, and 1% CaCO₃ in 96-well microtiter plates.The microtiter plates were incubated at 30° C. for one day withoutshaking. One microliter of each of the resulting reaction mixtures wasanalyzed for L-ascorbic acid formation using ascorbic acid test stripsand the RQFlex2 instrument (Merck KGaA, 64271 Darmstadt, Germany). Thepositive control strain was G. oxydans N44-1 grown under identicalconditions. By this method, the L-ascorbic acid-non-producing mutantN44-1-6A9 was identified. Southern blot hybridization analysis was thenperformed to confirm the presence of Tn5 in the chromosomal DNA ofmutant N44-1-6A9. 2 μg of chromosomal DNA isolated from the mutant wasdigested with either ApaI, ClaI, EcoRI, EcoRV, KpnI, StuI, BamHI, SalI,or HindIII and subjected to agarose gel electrophoresis (0.8% agarose).The gel was then treated with 0.25 N HCl for 15 min, followed by 0.5 NNaOH for 30 min. The DNA was transferred to a nylon membrane with therapid downward transfer system TurboBlotter (Schleicher & Schuell GmbH,Germany). The probe was prepared with PCR-DIG labeling kit (RocheDiagnostics GmbH, 68298 Mannheim, Germany) using primers Tn2419 (SEQ IDNO:3) and Tn3125R (SEQ ID NO:4) with plasmid pSUP2021 as the template. A707-bp PCR product was obtained.

The hybridization conditions used were as follows: hybridization wasdone under stringent hybridization conditions, e.g., 2 hours to 4 daysincubation at 42° C. using a digoxigenin (DIG)-labeled DNA probe(prepared by using a DIG labeling system; Roche Diagnostics GmbH, 68298Mannheim, Germany) in DigEasyHyb solution (Roche Diagnostics) with 100μg/ml salmon sperm DNA, followed by washing the filters for 15 min(twice) in 2×SSC and 0.1% SDS at room temperature and then washing for15 min (twice) in 0.5×SSC and 0.1% SDS or 0.1×SSC and 0.1% SDS at 65° C.

Following the hybridization step, the detection of hybridization wasdone with anti-DIG-AP conjugate (Roche Diagnostics GmbH, 68298 Mannheim,Germany) and ECF substrate (Amersham Biosciences Uppsala, Sweden) usingthe STORM instrument (Amersham Biosciences). All operations were doneaccording to the instructions of the suppliers.

Using the methods described above, the presence of Tn5 in the chromosomeof mutant N44-1-6A9 was confirmed.

3. Cloning of a DNA Fragment Interrupted by Tn5 and Sequencing of theAdjacent Regions.

Based on the results of the restriction enzyme digestions described insection 2 above, ApaI, ClaI, EcoRI, and EcoRV were selected as theenzymes that generated DNA fragments having more than 1 kb of flankingchromosomal DNA at both sides of the Tn5 insertion. Double digestion ofmutant N44-1-6A9 DNA with SalI (which cuts approximately in the middleof Tn5) and ApaI gave two fragments (6.2 and 3.8 kb) that hybridized tothe 707-bp probe described above.

The chromosomal DNA of the Tn5 mutant G. oxydans N44-1-6A9 was preparedand digested with ApaI. The DNA fragments with the size of from 9 to 12kb were isolated from agarose gel and ligated with the cloning vectorpBluescript II KS+ (Stratagene, Switzerland), previously digested withApaI. The ligation mixture was then used to transform competent E. colicells, selecting on L-agar plates containing 50 μg/ml kanamycin and 100μg/ml ampicillin. From one transformant, the plasmid was extracted andthe cloned regions flanking the Tn5 insertion were sequenced. Thenucleotide sequence of the single open reading frame (interrupted by theTn5 insertion) was assembled after removing a 9-bp duplication that isknown to occur during the Tn5 transposition event. The nucleotidesequence of the full open reading frame, hereafter referred to as theSNDHai gene of Gluconobacter oxydans N44-1, consisted of 2367 by and isgiven as SEQ ID NO:1. The corresponding amino acid sequence deduced fromthe nucleotide sequence of SEQ ID NO:1 is given here as SEQ ID NO:2.

The nucleotide sequence of the SNDHai gene (SEQ ID NO:1) was subjectedto a Blast 2 search (version 2 of BLAST from the National Center forBiotechnology Information [NCBI] described in Altschul et al. (NucleicAcids Res. 25: 3389-3402, 1997)) on the database PRO SW-SwissProt (fullrelease plus incremental updates). The conditions used were the gappedalignment and filtration of the query sequence for the low complexityregion.

Using the program MOTIFS, bacterial quinoprotein dehydrogenasessignature sequences were readily identified, indicating that SNDHai hasthe characteristics of a PQQ-dependent enzyme.

EXAMPLE 6 Southern Blot Analysis of the Bacteria Producing L-AscorbicAcid from L-Sorbosone

Chromosomal DNA was prepared from cells of Gluconobacter oxydans IFO3293, IFO 3292, IFO 3244, IFO 3287, Gluconobacter frateurii IFO 3260 andIFO 3265, Gluconobacter cerinus IFO 3266 and IFO 3269, Acetobacter acetisubsp. orleanus IFO 3259, Acetobacter aceti subsp. xylinum IFO 13693 andIFO 13773, Acetobacter sp. ATCC 15164, and Escherichia coli K-12.Strains IFO 13693 and IFO 13773 were grown at 27° C. for 3 days on No.350 medium containing 5 g/l Bactopeptone (Difco), 5 g/L yeast extract(Difco), 5 g/L glucose, 5 g/L mannitol, 1 g/L MgSO₄.7H₂O, 5 ml/Lethanol, and 15 g/L agar. All other Acetobacter strains and allGluconobacter strains were grown at 27° C. for 3 days on mannitol broth(MB) agar medium containing 25 g/l mannitol, 5 g/l yeast extract (DifcoLaboratories, Detroit, Mich., USA), 3 g/l Bactopeptone (Difco), and 18g/l of agar (Difco). E. coli K-12 was grown on Luria Broth agar medium.The chromosomal DNA preparations were used for Southern blothybridization under stringent conditions as described in Example 5. Thechromosomal DNA preparations were digested with ClaI (when analyzing theN-domain region) or EcoRI (when analyzing the C-domain region), and 1 μgof the DNA fragments were separated by agarose gel electrophoresis (1%agarose). The gel was treated with 0.25 N HCl for 15 min and then 0.5 NNaOH for 30 min, and then was blotted onto a nylon membrane with VacuumBlotter Model 785 (BIO-RAD Laboratories AG, Switzerland) according tothe instruction of the supplier. The probes were prepared with PCR-DIGlabeling kit (Roche Diagnostics) by using the primer sets as describedin Table 2. The PCR product P1 corresponds to the region of SNDHaidesignated the N-domain (possible transmembrane region) while PCRproduct P2 corresponds to the region of SNDHai designated as theC-domain (possible primary dehydrogenase region).

TABLE 2 Primers used for PCR to generate labeled probes for Southernhybridizations Expected size of SEQ ID NOs. PCR PCR product Primer setof primers product (bp) SNDH1F and SNDH420R 5 and 6 P1 420 SNDH501F andSNDH2364R 7 and 8 P2 1864 SNDH501F and SNDH1530R 7 and 9 P3 1030SNDH1391F and SNDH2364R 10 and 8  P4 974

Table 2 shows the results of the Southern blot hybridizationexperiments. In the hybridization with the P1 (N-domain) probe, clearpositive bands were observed for G. oxydans IFO 3293, IFO 3292, IFO3244, IFO 3287 and A. sp. ATCC 15164. In the hybridization with the P2(C-domain) probe, clear positive bands were observed for strains IFO3293, IFO 3292, IFO 3244, IFO 3287 and A. sp. ATCC 15164, while a faintband was observed for stains IFO 3260, IFO 3265, IFO 3266, IFO 3269 andIFO 13773. The control strain, E. coli K-12, showed no detectablesignals for either domain.

TABLE 3 Detection of hybridization signals in different strains obtainedby Southern blot hybridization with probes for N- and C-domains ofSNDHai (probes P1 and P2) Strain P1 P2 G. oxydans IFO 3293 + + G.oxydans IFO 3292 + + G. oxydans IFO 3244 + + G. frateurii IFO 3260 nd trG. frateurii IFO 3265 nd tr G. cerinus IFO 3266 nd tr G. oxydans IFO3269 nd tr G. oxydans IFO 3287 + + A. aceti subsp. orleanus IFO 3259 ndnd A. aceti subsp. xylinum IFO 13693 nd nd A. aceti subsp. xylinum IFO13773 nd tr Acetobacter sp. ATCC 15164 + + E. coli K-12 nd nd Tr, trace;nd, not detected. Probes P1 and P2 were synthesized (as DIG-labeled PCRproducts) with the primer sets specified in Table 2.

EXAMPLE 7 PCR Amplification and Sequencing of Orthologs of theGluconobacter Oxydans N44-1 SNDHai Gene

Chromosomal DNA preparations (prepared as described in Example 6) wereused as templates for PCR with the four primer sets shown in Table 2.Five to 100 ng of chromosomal DNA was used per reaction (total volume,50 μl). Unless specified otherwise, the Expand High Fidelity PCR systemwas used (Roche Diagnostics). The PCR conditions were as follows:

Incubation at 94° C. for 2 min; 30 cycles of (i) denaturation step at94° C. for 15 sec, (ii) annealing step at 60° C. for 30 sec, (iii)synthesis step at 72° C. for 45 to 120 sec (time for the synthesis stepfor primer sets P1, P2, P3 and P4 were 45 sec, 120 sec, 90 sec, and 90sec, respectively); extension at 72° C. for 7 min.

Samples of the PCR reactions were separated by agarose gelelectrophoresis and the bands were visualized with a transilluminatorafter staining with ethidium bromide. The results of the PCR reactionsare summarized in Table 4.

TABLE 4 Detection of PCR products P1, P2, P3 and P4 in different strainsobtained with the primer sets of Table 2 (products visualized viaagarose gel electrophoresis) Strain P1 P2 P3 P4 G. oxydans IFO 3293 + +*nt + G. oxydans IFO 3292 + nd nd + G. oxydans IFO 3244 + + + + G.frateurii IFO 3260 nd nd nd nd G. cerinus IFO 3266 nd nd nd nd G.oxydans IFO 3287 + + nd + A. aceti subsp. orleanus IFO 3259 nd nd nd ndA. aceti subsp. xylinum IFO 13693 nd nd nd nd A. aceti subsp. xylinumIFO 13773 nd nd nd nd Acetobacter sp. ATCC 15164 + + nd nd E. coli K-12nd nd nt nd +, detected; nd, not detected; nt, not tested. *This PCR wasdone with GC-rich PCR system (Roche Diagnostics) with the same reactioncycle as was used for Expand High Fidelity PCR system.

When clear PCR bands were observed on the agarose gel (Table 4), the PCRproducts were used directly for nucleotide sequencing using standardmethods. The nucleotide sequences obtained for the different PCRproducts, and the corresponding amino acid sequences of the encodedpeptides, were compared with the full length sequence of the SNDHai geneand protein from G. oxydans N44-1.

Gluconobacter oxydans IFO 3292 SNDHai Ortholog

The PCR product (about 1 kb) obtained upon amplification with primersSNDH1391F (SEQ ID NO:10) and SNDH2364R (SEQ ID NO:8) and chromosomal DNAfrom G. oxydans IFO 3292 as the template, was used for sequencing withprimer SNDH1391F (SEQ ID NO:10). The determined nucleotide sequence of771 by (SEQ ID NO:11) showed 98.7% (761/771) homology with nucleotides1431-2201 of the sequence of SNDHai fromG. oxydans N44-1 (SEQ ID NO:1).The deduced amino acid sequence of 256 amino acids (SEQ ID NO:12) showed100% identity to amino acids 478-733 of the amino acid sequence of SNDHfrom G. oxydans N44-1 (SEQ ID NO:2).

Gluconobacter oxydans IFO 3287 SNDHai Ortholog

The PCR product (about 0.4 kb) obtained upon amplification with primersSNDH1F (SEQ ID NO:5) and SNDH420R (SEQ ID NO:6) and chromosomal DNA fromG. oxydans IFO 3287 as the template, was used for sequencing with primerSNDH420R (SEQ ID NO:6). The determined nucleotide sequence of 350 by(SEQ ID NO:13) showed 97.4% (341/350) homology with nucleotides 31-380of SEQ ID NO:1. The deduced amino acid sequence of 116 residues (SEQ IDNO:14) showed 100% identity with amino acids 11-126 of SEQ ID NO:2.

The PCR product (about 1.9 kb) obtained upon amplification with primersSNDH501F (SEQ ID NO:7) and SNDH2364R (SEQ ID NO:8) was used forsequencing with primer SNDH501F (SEQ ID NO:7). The determined nucleotidesequence of 808 by (SEQ ID NO:15) showed 98.0% (745/808) homology withnucleotides 578-1385 of SEQ ID NO:1). The deduced amino acid sequence of268 residues (SEQ ID NO:16) showed 100% identity to amino acids 194-461of SEQ ID NO:2.

The PCR product (about 1 kb) obtained upon amplification with primersSNDH1391F (SEQ ID NO:10) and SNDH2364R (SEQ ID NO:8) was used forsequencing with primer SNDH1391F (SEQ ID NO:10). The determinednucleotide sequence of 800 by (SEQ ID NO:17) showed 98.8% (790/800)homology with nucleotides 1469-2268 of SEQ ID NO:1. The deduced aminoacid sequence of 266 residues (SEQ ID NO:18) showed 100% identity withamino acids 491-756 of SEQ ID NO:2.

Acetobacter sp. ATCC 15164 SNDHai Ortholog

The PCR product (about 0.4 kb) obtained upon amplification with primersSNDH1F (SEQ ID NO:5) and SNDH420R (SEQ ID NO:6) and chromosomal DNA fromA. sp. ATCC 15164 as the template, was used for sequencing with primerSNDH420R (SEQ ID NO:6). The determined nucleotide sequence of 360 by(SEQ ID NO:19) showed 97.8% (352/360) homology with nucleotides 31-390of SEQ ID NO:1. The deduced amino acid sequence of 120 residues (SEQ IDNO:20) showed 100% identity with amino acids 11-130 of SEQ ID NO:2.

The PCR product (about 1.9 kb) obtained upon amplification with primersSNDH501F (SEQ ID NO:7) and SNDH2364R (SEQ ID NO:8) was used forsequencing with primer SNDH501F (SEQ ID NO:7). The determined nucleotidesequence of 760 by (SEQ ID NO:21) showed 98.0% (745/760) homology withnucleotides 563-1322 of SEQ ID NO:1. The deduced amino acid sequence of252 residues (SEQ ID NO:22) showed 100% identity with amino acids189-440 of SEQ ID NO:2.

Gluconobacter oxydans IFO 3244 SNDHai Ortholog

Complete nucleotide sequence of the SNDHai ortholog gene of G. oxydansIFO 3244 was determined by using the PCR products obtained with thechromosomal DNA of G. oxydans IFO 3244 as the template and the followingprimer sets: SNDH1F (SEQ ID NO:5) and SNDH420R (SEQ ID NO:6); SNDH501F(SEQ ID NO:7) and SNDH1530R (SEQ ID NO:9); SNDH1391F (SEQ ID NO:10) andSNDH2364R (SEQ ID NO:8); SNDH382 (SEQ ID NO:23) and SNDH1530R (SEQ IDNO:9); SNDH1F (SEQ ID NO:5) and SNDH689R (SEQ ID NO:24). Chromosomal DNAdigested with BglII and BamHI and ligated was used for two more PCRswith following primer sets: SNDH420R (SEQ ID NO:6) and SNDH501F (SEQ IDNO:7) and SNDH1530R (SEQ ID NO:9) and IS-50.3 (SEQ ID NO:25). Thecomplete nucleotide sequence (SEQ ID NO:26) showed 98.4% homology to thenucleotide sequence of SNDHai from G. oxydans N44-1 (SEQ ID NO:1). Thededuced amino acid sequence (SEQ ID NO:27) showed 100% identity to theamino acid sequence of SEQ ID NO:2.

EXAMPLE 8 Increased L-ascorbic Acid Production from L-sorbosone byIncreasing the SNDHai Gene Dosage

The SNDHai gene with upstream and downstream flanking regions wasamplified by PCR with chromosomal DNA of strain N44-1 as template andthe primer set N1 (SEQ ID NO:28) and N2 (SEQ ID NO:29).

The PCR was done with the GC-rich PCR system (Roche Diagnostics)according to the instructions of the supplier. The amplified DNAfragment was inserted into vector pCR2.1-TOPO (Invitrogen, Carlsbad,Calif., USA). The resulting plasmid was then digested with HindIII andXhoI. The HindIII-XhoI fragment including the SNDHai gene was ligated tovector pVK100 (available from the American Type Culture Collection,catalog no. ATCC 37156) previously treated with HindIII and XhoI. Theligation mixture was used to transform E. coli TG1. The desired plasmid,designated pVK-P-SNDHai-T, was isolated from E. coli, and introducedinto G. oxydans strain N44-1 by electroporation using standard methods(Electrocell manipulator ECM600, BTX Inc., San Diego, Calif., USA).

Cells of G. oxydans strains N44-1 and N44-1 carrying the plasmidpVK-P-SNDHai-T were cultivated in 50 ml of No. 5 medium containing 100g/l D-sorbitol, 0.5 g/l glycerol, 15 g/l yeast extract (Difco), 2.5 g/lof MgSO₄.7H₂O, and 15 g/L of CaCO₃ in a 500 ml baffled shake flask at30° C. with shaking at 200 rpm. After 48 h of cultivation, the amountsof L-ascorbic acid measured in the supernatant by HPLC in the two flaskswere 110 mg/l and 200 mg/l, respectively.

EXAMPLE 9 Production of L-ascorbic Acid from L-sorbosone using RestingCells Grown on Mannitol Broth Agar Medium

IFO strains 3293, 3292, 3244, 3260, 3266, 3287, 3259, 13693, and 13773as well as Acetobacter sp. ATCC 15164 and Gluconobacter oxydans N44-1, aderivative of the strain IFO 3293, were used for the production ofL-ascorbic acid from L-sorbosone.

Strains IFO 13693 and IFO 13773 were grown at 27° C. for 3 days on No.350 medium containing 5 g/l Bactopeptone (Difco), 5 g/l yeast extract(Difco), 5 g/l glucose, 5 g/l mannitol, 1 g/l MgSO₄.7H₂O, 5 ml/lethanol, and 15 g/l agar. All other Acetobacter strains and allGluconobacter strains were grown at 27° C. for 3 days on mannitol broth(MB) agar medium containing 25 g/l mannitol, 5 g/l yeast extract (DifcoLaboratories, Detroit, Mich., USA), 3 g/l Bactopeptone (Difco), and 18g/l of agar (Difco).

Cells were scraped from the agar plates, suspended in distilled waterand used for resting cell reactions conducted at 30° C. for 20 h in 5 mltubes with shaking at 230 rpm. The reaction mixtures (0.5 ml) contained1% L-sorbosone, 0.3% NaCl, 1% CaCO₃ and cells at a final concentrationof 10 absorbance units at 600 nanometers (OD₆₀₀). At the conclusion ofthe incubation period, the reaction mixtures were analyzed by highperformance liquid chromatography (HPLC) using an Agilent 1100 HPLCsystem (Agilent Technologies, Wilmington, USA) with aLiChrospher-100-RP18 (125×4.6 mm) column (Merck, Darmstadt, Germany)attached to an Aminex-HPX-78H (300×7.8 mm) column (Biorad, Reinach,Switzerland). The mobile phase was 0.004 M sulfuric acid, and the flowrate was 0.6 ml/min. Two signals were recorded using an UV detector(wavelength 254 nm) in combination with a refractive index detector. Inaddition, the identification of the L-ascorbic acid was done using anamino-column (YMC-Pack Polyamine-II, YMC, Inc., Kyoto, Japan) with UVdetection at 254 nm. The mobile phase was 50 mM NH₄H₂PO₄ andacetonitrile (40:60).

An Agilent Series 1100 HPLC-mass spectrometry (MS) system was used toidentify L-ascorbic acid. The MS was operated in positive ion mode usingthe electrospray interface. The separation was carried out using aLUNA-C8(2) column (100×4.6 mm) (Phenomenex, Torrance, USA). The mobilephase was a mixture of 0.1% formic acid and methanol (96:4). L-Ascorbicacid eluted with a retention time of 3.1 minutes. The identity of theL-ascorbic acid was confirmed by retention time and the molecular massof the compound.

To exclude the presence of D-isoascorbic acid, the identification ofL-ascorbic acid was additionally done by retention time using anamino-column (YMC-Pack Polyamine-II, YMC, Inc., Kyoto, Japan) with UVdetection at 254 nm. The mobile phase was 50 mM NH₄H₂PO₄ andacetonitrile (40:60).

The Gluconobacter and Acetobacter strains produced L-ascorbic acid fromL-sorbosone as shown in Table 5.

TABLE 5 Production of L-ascorbic acid from L-sorbosone L-ascorbic acidStrain (mg/L) G. oxydans IFO 3293 1740 G. oxydans N44-1 570 G. oxydansIFO 3292 410 G. oxydans IFO 3244 1280 G. frateurii IFO 3260 50 G.cerinus IFO 3266 140 G. oxydans IFO 3287 60 A. aceti subsp. Orleanus IFO3259 30 A. aceti subsp. Xylinum IFO 13693 40 A. aceti subsp. Xylinum IFO13693 120 Acetobacter sp. ATCC 15164 310 Blank Not detected Blank;reaction was done in the reaction mixture without cells.

EXAMPLE 10 Production of L-ascorbic Acid from D-sorbitol, L-sorbose orL-sorbosone using Resting Cells Grown on 3BD Agar Medium

Cells of G. oxydans N44-1 were grown at 27° C. for 3 days on No. 3BDagar medium containing 70 g/l L-sorbose, 0.5 g/l glycerol, 7.5 g/l yeastextract (Difco), 2.5 g/l MgSO₄.7H₂O, 10 g/l CaCO₃ and 18 g/l agar(Difco). The resting cell reactions (1 ml reaction mixture in 10 mltube) were carried out with 2% D-sorbitol, 2% L-sorbose, or 1%L-sorbosone at 30° C. for 24 h as described in Example 9. Strain N44-1produced 280, 400 and 1780 mg/l of L-ascorbic acid from D-sorbitol,L-sorbose, and L-sorbosone, respectively.

Other reactions (0.5 ml reaction mixture in 10 ml tube) were carried outwith N44-1 cells grown on No. 3BD agar medium in reaction mixturescontaining 2% D-sorbitol, 2% L-sorbose or 2% L-sorbosone for 2 days asdescribed in Example 9. Strain N44-1 produced 1.8, 2.0 and 5.1 g/l ofL-ascorbic acid from D-sorbitol, L-sorbose, and L-sorbosone,respectively.

A reaction using cells of G. oxydans IFO 3293 was carried out with 2%L-sorbosone as described above. Strain IFO 3293 produced 5.7 g/l ofL-ascorbic acid in 2 days.

EXAMPLE 11 Production of L-ascorbic Acid from D-sorbitol using RestingCells Grown in Liquid Medium

Cells of G. oxydans N44-1 were grown in 200 ml of No. 5 mediumcontaining 100 g/l D-sorbitol, 0.5 g/l glycerol, 15 g/l yeast extract(Fluka BioChemika, Buchs, Switzerland), 2.5 g/l MgSO₄.7H₂O and 15 g/lCaCO₃ in a 2-l baffled shake flask at 30° C. with shaking at 180 rpm.After 24 h, the culture was centrifuged at 3220 g (Eppendorf 5810R,Hamburg, Germany), and the cells were resuspended in 0.9% NaCl solution,centrifuged again at 3220 g and the cell pellet was used to inoculateone baffled 500 ml shake flask containing 50 ml of full growth medium(100 g/l D-sorbitol, 0.5 g/l glycerol, 15 g/l yeast extract, 2.5 g/lMgSO₄.7H₂O, 15 g/l CaCO₃) and another baffled 500 ml shake flaskcontaining 50 ml production medium (100 g/l D-sorbitol, 3 g/l NaCl, 10g/l CaCO₃). The initial cell density, measured as optical density at 600nm (OD₆₀₀), in both flasks was 10. Both flasks were incubated at 30° C.with shaking at 180 rpm. After 48 h, the cell suspension in growthmedium and production medium had accumulated 1.06 and 1.18 g/lL-ascorbic acid, respectively. No additional growth was observed in fullmedium during the incubation period time.

EXAMPLE 12 Production of L-ascorbic Acid from L-sorbosone or D-sorbitolby Resting Cells of Recombinant Microorganisms with Increased SNDHaiGene Dosage

The SNDHai gene of G. oxydans N44-1 (SEQ ID NO:1) with upstream anddownstream flanking regions was amplified by PCR with chromosomal DNA ofstrain N44-1 as template and the primer set N1 (SEQ ID NO:28) and N2(SEQ ID NO:29).

The PCR was done with the GC-rich PCR system (Roche Diagnostics GmbH)according to the instructions of the supplier. The amplified DNAfragment was inserted into vector pCR2.1-TOPO (Invitrogen, Carlsbad,Calif., USA). The resulting plasmid was then digested with HindIII andXhoI. The HindIII-XhoI fragment including the SNDHai gene was ligated tovector pVK100 (available from the American Type Culture Collection,catalog no. ATCC 37156) previously treated with HindIII and XhoI. Theligation mixture was used to transform E. coli TG1. The desired plasmid,designated pVK-P-SNDHai-T, was isolated from E. coli, and introducedinto G. oxydans strain N44-1 by electroporation using standard methods(Electrocell manipulator ECM600, BTX Inc., San Diego, Calif., USA).

Three independent transformants, designated N44-1(pVK-P-SNDHai-T) clonenumber 1, 2, and 3, together with the parental strain G. oxydans N44-1,were each grown on No. 3BD agar and MB agar media. The cells werescraped from the plates and used for resting cell reactions (1%L-sorbosone as the substrate) as described in Example 9. After growth onNo. 3BD agar, in the resting cell assay strain N44-1 produced 2.5 g/lL-ascorbic acid, while strains N44-1(pVK-P-SNDHai-T) clones 1, 2 and 3produced 4.2, 4.1 and 4.2 g/l L-ascorbic acid, respectively. Aftergrowth on MB agar, in the resting cell assay strain N44-1 produced 0.12g/l L-ascorbic acid, while strains N44-1(pVK-P-SNDHai-T) clones 1, 2 and3 produced 1.8, 2.5 and 0.94 g/l L-ascorbic acid, respectively.

Another reaction was carried out using cells of G. oxydans N44-1 andclone 2 (see above) cultivated in 50 ml of No. 5 medium (100 g/lD-sorbitol, 0.5 g/l glycerol, 15 g/l yeast extract, 2.5 g/lMgSO₄.7H₂O,15 g/l CaCO₃) in duplicate 500 ml baffled shake flasks at 30° C. withshaking at 220 rpm for 3 days. From one flask for each strain, theresulting broth was centrifuged at 500 rpm to remove CaCO₃. Thesupernatant from this step was then centrifuged at 5,000 rpm to pelletthe cells. The collected cells were re-suspended in 10 ml of 0.9% NaClsolution, and again centrifuged at 5,000 rpm to pellet the cells. Thecollected cells were re-suspended in water and used to inoculate 1 ml ofproduction medium (20 g/l D-sorbitol, 3 g/l NaCl, 10 g/l CaCO₃) in 10 mlreaction tube at a final resting cell density corresponding to 5 ODunits at 600 nm. After 20 h reaction time at 30° C. and 220 rpm, thesupernatant harvested from the production flask contained 360 and 760mg/l L-ascorbic acid, respectively for strains N44-1 and N44-1overexpressing SNDHai. In contrast, after 72 h the supernatant harvestedfrom the remaining growth medium contained 0 and 440 mg/l L-ascorbicacid, respectively.

EXAMPLE 13 Production of L-ascorbic Acid from L-sorbosone in RestingCells of E. coli

The SNDHai gene without stop codon named SNDHai-1, corresponding tonucleotides 1-2364 of SEQ ID NO:1, was amplified from strain N44-1chromosomal DNA by PCR (Roche High Fidelity kit) using the primer pairSNDHai-Nde (SEQ ID NO:30) and SNDHaiHis-X (SEQ ID NO:31).

The amplified DNA was cloned into pCR2.1-TOPO (Invitrogen, Carlsbad,Calif., USA) to obtain pCR2.1-TOPO-SNDHai-1, whose SNDHai sequence wasconfirmed to be correct by nucleotide sequencing. Then the SNDHai-1 genewas cut out with NdeI and XhoI and ligated between NdeI and XhoI sitesof pET-21b(+) (Novagen, Madison, Wis., USA) to produce pET21b-SNDHaiHis;6×His was added at the C-terminus of SNDHai. The pET21b-SNDHaiHis wasintroduced into E. coli BL21 (DE3).

Five ml of one overnight culture of E. coli BL21 (DE3)/ pET21b-SNDHaiHisin LB with carbenicillin 50 μg/ml was inoculated into 200 ml of the samemedium. The cells were cultivated at 230 rpm at 37° C. for 2 h, theninduced with 1 mM IPTG and continued to be cultivated at 230 rpm at 25°C. for 3 h. The resulting culture was centrifuged and washed twice withsaline and the cell pellet was resuspended in 2 ml of water. The cellswere used for resting cell reaction with the reaction mixture (500 μl in5 ml tube) containing cells at OD600=10, 1% sorbosone monohydrate, 5 μMPQQ, 5 mM MgCl₂, 0.3% NaCl, and 1% CaCO₃ conducted at 30° C. for 15 h.0.14 g/L of L-ascorbic acid was produced after incubation for 15 h. Whenthe resting cell reaction was done with 1 μM PQQ (the other conditionswere same as those described above), 0.05 g/L of L-ascorbic acid wasproduced after incubation for 3 h.

EXAMPLE 14 Production of L-ascorbic Acid from D-sorbitol by RestingCells of Recombinant Microorganisms with Increased SNDHai Gene Dosage

Cells of G. oxydans N44-1 overexpressing SNDHai are grown in 50 ml ofNo. 5 medium containing 100 g/l D-sorbitol, 0.5 g/l glycerol, 15 g/lyeast extract (Fluka BioChemika, Buchs, Switzerland), 2.5 g/l MgSO₄.7H₂Oand 15 g/l CaCO₃ in a 500-ml baffled shake flask at 30° C. with shakingat 180 rpm for 48 h. The resulting cell suspension is used to inoculatea 2-L bioreactor, called growth vessel (Biostat-MD, B. Braun Melsungen,Melsungen, Germany) containing 1.25 l of medium composed of 100 g/lD-sorbitol, 15 g/l yeast extract (Fluka BioChemika, Buchs, Switzerland),2.5 g/l MgSO₄.7H₂O, 0.3 g/l KH₂PO₄ and 0.12 g/l CaSO₄. Cells arecultivated at 30° C., 1 l/min aeration rate, the pH is controlled to 5.7with a 25% solution of Na₂CO₃, dissolved oxygen is controlled to 10%saturation by varying the stirring speed. After 24 h, the cell densitymeasured as absorption units at 600 nm is 20. At this time point, a feedsolution containing 100 g/l D-sorbitol, 15 g/l yeast extract (FlukaBioChemika, Buchs, Switzerland), 2.5 g/l MgSO₄.7H₂O, 0.3 g/l KH₂PO₄ and0.12 g/l CaSO₄ is fed into the growth vessel at a feed rate of 125 ml/h,and broth is continuously harvested at a harvest rate of 125 ml/h. Bythis means, the volume in the growth vessel is kept constant at 1.25 l.Other process parameters continue to be controlled as mentioned above.

This broth is continuously fed at a rate of 125 ml/h into a secondreactor, called production vessel, filled with 5 l production mediumcontaining 100 g/l D-sorbitol, 0.3 g/l NaCl and 0.12 g/l CaSO₄, and thetemperature is kept at 30° C., pH at 7.0 by controlling with a 20%solution of NaOH. The aeration rate is kept constant at 10 l/min, anddissolved oxygen is controlled at 20% by varying the stirrer speed.Production medium with the same composition is also continuously fed tothe production vessel at a feed rate of 375 ml/h. The vessel volume iskept constant at 5 l by continuously harvesting supernatant at 500 ml/hrate, resulting as a filtrate stream from a crossflow ultrafiltrationmodule with 500 kDa pore size (UFP-500-E-9A, Amersham Biosciences),through which the cell suspension harvested from the production vesselis pumped at 50 l/h using a Masterflex pump. The retentate flow ispumped back into the vessel. Once the cell density in the productionvessel reaches 100 absorption units at 600 nm, cells start to beharvested from the concentrated cell stream flowing back into theproduction vessel at a rate of 25 ml/h, in order to keep the celldensity in the production vessel constant.

The harvest stream of cell-free supernatant contains 4 g/l L-ascorbicacid and is continuously fed at a rate of 500 ml/h into a collectingvessel with a double jacket at 30° C. (Ecoline Re112, Lauda,Lauda-Koenigshofen, Germany). This vessel feeds continuously supernatantto the diluate compartment of a two-compartment electrodialysis unit(stack containing 10 cell pairs with cation exchange membranes CMX-S andanion exchange membranes ASM, total membrane area 0.2 m², from EurodiaIndustries, Wissous, France) at a rate of 180 l/h, and a constant streamis pumped out of the vessel to keep its volume constant at 2 l. Anothervessel with double jacket containing initially deionized water at 30° C.is continuously fed with fresh deionized water at a rate of 62.5 ml/h,pumps constantly aqueous solution into the concentrate compartment ofthe electrodialysis unit at a rate of 200 l/h, and a constant harveststream is pumped out of the vessel. Feed solutions are pumped to theelectrodialysis stack using peristaltic pumps (7518-00, Masterflex,USA), and recirculation of solutions through each electrodialysiscompartment is done with help of rotary pumps (MD-20, IWAK, Tokyo,Japan). During the whole process, 14 V are applied to theelectrodialysis stack (power source FuMATech TS001/5, St. Ingbert,Germany). The concentration of L-ascorbic acid in the harvest stream is16 g/l.

EXAMPLE 15 Purification of L-ascorbic Acid Produced by a Resting CellReaction via Downstream Processing Steps

The harvest stream of Example 14 containing 16 g/l L-ascorbic acid isfed to a chelating resin (Amberlite IRC 748, Rohm and Haas,Philadelphia, Pa., USA) to eliminate divalent cations from the stream.It is then collected in a cooled vessel (feed vessel), and when 10 lhave been collected, they are processed in batch mode through a bipolarmembrane electrodialysis unit (stack containing 7 Neosepta BP1/CMBmembranes, total membrane area 0.14 m², from Eurodia Industries,Wissous, France). This solution is pumped at 200 l/h through the feedcompartment of the electrodialysis unit, and recycled into the feedvessel. Another cooled vessel (concentrate vessel) containing initially5 l of a 2 g/l NaOH solution is pumped at 100 l/h through theconcentrate compartment of the bipolar membrane electrodialysis unit. Byapplying a maximal voltage of 25 V and maximal electric current of 20 A,sodium cations from the feed compartment are transferred to theconcentrate compartment, and thus the sodium form of L-ascorbic acidpresent in the feed stream is converted into the corresponding free acidform. After reaching 90% conversion yield, the process is stopped. Inthe concentrate vessel, 6 l of solution containing 7.5 g/l NaOH arecollected in the diluate vessel, 9 l solution containing about 16 g/lL-ascorbic acid in its free acid form and 1.6 g/l L-ascorbic acid in itssodium salt form are further processed through a cation exchange resin(Amberlite FPC 21 H, Rohm and Haas, Philadelphia, Pa., USA), in order toincrease conversion yield of the sodium salt into the free acid form toabout 99%. Alternatively, the 10 1 solution containing 16 g/l L-ascorbicacid in its sodium salt form coming from the electrodialysis step isdirectly treated by cation exchange resin, being converted to the freeacid form at 99% yield. The stream of L-ascorbic acid in the form of thefree acid, obtained by either of the methods described above, is thenfurther processed by a sequence of the following steps: anion exchange,activated carbon treatment, concentration, crystallization, filtrationof the crystals, and drying. The final purity of the obtained crystalsis 98%, and the yield obtained with the combined downstream processingsteps is 80%.

1-42. (canceled)
 43. A process for the production of L-ascorbic acidcomprising: (a) culturing microorganism under conditions which enablegrowth; (b) changing the conditions such that the growth rate of themicroorganism is reduced, leading to resting cells; and (c) producingL-ascorbic acid from a substrate using the resting cells produced instep (b), wherein the substrate is selected from the group consisting ofD-glucose, D-sorbitol, L-sorbose, L-sorbosone, 2-keto-L-gulonate,D-gluconate, 2-keto-D-gluconate and 2,5-diketo-gluconate.
 44. Theprocess of claim 43, wherein the steps of culturing and producingL-ascorbic acid are performed in 2 or more separate vessels.
 45. Theprocess of claim 43, wherein the steps of culturing and producingL-ascorbic acid are not separated by any step of washing, any step ofisolating, or both.
 46. The process of claim 43, wherein the step ofproducing L-ascorbic acid is performed in batch, fed-batch, continuous,or a semi-continuous mode.
 47. The process of claim 43, wherein themicroorganism is grown in batch, fed-batch, continuous, or asemi-continuous mode.
 48. The process of claim 43, wherein the densityof the resting cells in the medium measured as OD at 600 nm is at least10.
 49. The process of claim 43, wherein the yield of producedL-ascorbic acid is at least 1.8 g/l.
 50. The process of claim 43,wherein the microorganism is selected from the group consisting ofyeast, algae, and bacteria.
 51. The process of claim 43, wherein themicroorganism is selected from the group consisting of Candida,Saccharomyces, Zygosaccharomyces, Scyzosaccharomyces, Kluyveromyces,Chlorella, Gluconobacter, Acetobacter aceti, Pantoea, Cryptococcus,Pseudomonas and Escherichia.
 52. The process of claim 43, wherein theprocess uses a microorganism capable of producing both L-ascorbic acidand 2-keto-L-gulonic acid from a substrate and wherein the ratio betweenthe concentration of the L-ascorbic acid and the 2-keto-L-gulonic acidis more than 0.1.
 53. The process of claim 43, further comprising:separating L-ascorbic acid from components in the medium usingelectrodialysis.
 54. The process of claim 43, further comprising:isolating L-ascorbic acid from the medium; and optionally, performingone or more additional purification steps.